© 1991 S. Karger AG. Basel 0028 2766/91 0581 -0075S2.75/0
Nephron 1991;58:75-84
Isolation and Characterization of Chicken Mesangial Cells1 Marc J. Sadovnica, Jessica Brand-Elnaggarb, W. Kline Boltonb a Division of Nephrology. Alleghany General Hospital, Pittsburgh, Pa, and l>Division of Nephrology, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Va., USA
Key Words. Mesangial cells • Cell culture, chicken • Non-mammalian, cell culture
Introduction Glomerulonephritis in man is associated with prolif eration of endogenous glomerular cells and an influx of exogenous inflammatory cells [1. 2]. The resultant histo logic lesions consist of variable degrees of glomerular tuft hypercellularity, at times associated with extracapillary proliferation. Anti-glomerular basement membrane (GBM) disease comprises 5% of all biopsies in patients with glomerulonephritis; however, approximately 25-30% of patients with a more malignant form of glo merulonephritis associated with rapidly progressive re nal failure (RPGN) have anti-GBM disease [1, 2]. An 1 This work was supported by USPHS grant DK 32530 from the NID D K .and NRSAgrant numbersT32 AM 07156-11 and5T32 HL 07284-10.
additional large subsegment of idiopathic RPGN con sists of those patients with minimal or no immune depos its, but with identical lesions by light microscopy [3]. Much evidence suggests a role for cell-mediated immu nity in many experimental models and in numerous types of glomerulonephritis in man [4, 5]. Unfortunately, few species lend themselves to the study of the role of cellmediated immunity in the pathogenesis of autoimmune glomerulonephritis. The model which most closely ap proximates anti-GBM human disease, Steblay nephritis, is induced by immunization of animals with heterologous GBM antigen [6], However, of mammalian species, only sheep and goats develop significant proliferative glomer ulonephritis, at times with crescents [6,7], GBM immuni zation of mice, rats, rabbits, guinea pigs, and monkeys results in either no detectable proliferation within glom eruli or variable proliferative changes (monkeys) [8-16].
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Abstract. Experimental autoimmune glomerulonephritis (EAG) in chickens appears to be mediated by cellular immunity and is associated with mesangial proliferation. We have developed techniques for the culture of chicken mesangial cells to study factors in vitro which lead to this proliferation. Chicken glomeruli isolated by sieving collagenase-treated whole kidney homogenates were cultured in Waymouth’s medium MB 752/1 supplemented with 20%decomplemented fetal calf serum and 1unit/ml insulin. Propagated cells share the following characteristics with mammalian mesangial cells: stellate and spindle-shaped morphology with an extensive microfilamentous system by light and electron microscopy; resistance to aminonucleoside of puromvein: susceptibility to mitomycin C : growth in L-valine-free medium; absent staining for factor Vlll-related antigen, chicken Tcell and la antigen: positive staining for fibronectin, myosin, a-actinin and desmin, and angiotensin II binding and induction of contraction. Unlike cultured mammalian mesangial cells, chicken mesangial cells avidly phagocytize latex beads and display multilamelIar residual bodies on electron microscopy indicative of phagocytic activity. They differed from fibroblasts which were non-phagocytic, had different growth patterns, fluorescence staining and ultrastructural morphology. To our knowledge, this is the first description of the culture of chicken mesangial cells. This in vitro system should allow further studies of pathogenetic processes involved in the production of EAG with elucidation of mechanisms relevant to human disease.
Sadov n ie /Brand-El nuggar/Bolton
Thus, despite immunization with GBM antigen and the development of antibodies to basement membrane, most mammalian species do not develop a proliferative glo merulonephritis. It is therefore difficult to examine the role of cell-mediated immunity in the pathogenesis of autoimmune glomerulonephritis in the absence of a pro liferative model in mammals. For these reasons we ex plored the development of autoimmune glomerulone phritis in chickens. We have reported that experimental autoimmune glomerulonephritis (EAG) can be induced in chickens by immunization with GBM antigen [17]. Normal chickens and birds made humorally incompetent through chemical bursectomy, then immunized with glomeruli, develop proliferative glomerulonephritis [18, 19]. Crescents are present in 3-28% of glomeruli depend ing on the strain and type of antigen utilized for immuni zation. EAG appears unrelated to the presence or ab sence of antibody bound in glomeruli and may be trans ferred to other chickens by mononuclear cells, but not by antibody [20]. Proliferation of mesangial cells is a major component of the hypercellularity in the glomerulone phritis in this model [19, 20], Since cell-mediated immu nity appears to be involved in the development of this mesangial hypercellularity in vivo, it would seem appro priate to examine the effect of various components of the cellular system in vitro with chicken mesangial cells. Although an extensive literature is available describing culture of mammalian mesangial cells, there is no infor mation available about culturing chicken mesangial cells. The purpose of the present report is to describe the methodology and techniques that we have developed to culture chicken mesangial cells and to characterize these cells in culture. Methods Glomerular Isolation Preparations of mammalian glomeruli predominantly free of tubular contamination and capsules may be obtained by passing cortical tissue through a series of graded metal sieves [21]. Several factors complicate the isolation of chicken glomeruli. The avian kidney lacks a well-demarcated cortex separating glomeruli from other renal tissue [20, 22]. Glomeruli are widely distributed in size with diameters ranging from 5 to 20 um (reptilian vs. mammalian), and small glomeruli are comparable in size to proximal tubules. Sieving of chicken kidney results in glomerular preparations heavily contaminated by tubules. In addition, this process fails to strip Bowman's capsule. Unencapsulated glomeruli are required for out growth in the chicken, as in other species [23]. To circumvent these difficulties, we explored a variety of techniques. The following procedure results in a preparation of unencapsulated glomeruli more than 80% free of tubular contamination.
Both kidneys are harvested aseptically from a single chicken, stripped of capsule and surrounding non-parenchymal tissue with forceps, then placed in a small tissue blender(Eberbach, Ann Arbor. Mich.) with ice-cold phosphate-buffered saline (PBS) to a final volume of 20 ml. Homogenization is carried out in six 15-second bursts separated by 15-second intervals to prevent tissue from over heating. The resultant mixture is added to a solution of 80 mg type-1 collagenase (Sigma. St. Louis, Mo.) in 50 ml of PBS which has been sterilized by passage through a 0,2-gm filter. Digestion is allowed to proceed at room temperature for 15 min with vigorous stirring. The resultant slurry is poured on top of four stacked 3-inch metal sieves (Newark Wire Cloth Co., Newark, N.J.) of mesh (upper to lower) No. 25. No. 140, No. 200, and No. 400 (opening size 710,107.74, and 38 gm:. respectively) resting on a beaker to catch the filtrate. Tissue is pressed through the upper sieves using a 25-ml Erlenmeyer flask as a pestle and washing with cold PBS forced from a syringe. Tissue caught on the No. 400 mesh is washed off with 30-40 ml of ice-cold PBS into a beaker and centrifuged at 400 g for 10 min. The supernat ant is discarded. Pellets are resuspended in culture medium and pooled. The final preparation consists primarily of unencapsulated glomeruli. Glomerular purity was 85% as previously described [20]. Resuspended glomeruli are distributed among 75-cmJ culture flasks (Costar. Cambridge, Mass.) with 25 ml of medium and placed in a humidified incubator with 5% CO» at 30°C. Glomerular Culture Multiple sera (fetal calf, chicken, goat, lamb) and culture media (Waymouth's MB 752/1, Dulbecco's modified Eagle's medium, minimum essential medium [MEM], RPMI 1640) were investigated. Outgrowth was most rapid with Waymouth’s medium MB 752/1 supplemented with 20% decomplemented fetal calf serum (FC'S), I unit/ml insulin, and antibiotics (standard medium): penicillin 100 units/ml, streptomycin 100 ug/ml, and amphotericin B 0.25 pg/ml (all media components :Gibco. Grand Island. N.Y.). Outgrowth and proliferation were favored by higher serum concentration. FCS proved better than goat serum and both were superior to lamb and chicken sera. Waymouth's medium provided a slight advantage over RPMI. Dulbecco's modified Eagle’s medium, and MEM resulted in poor mesangial cell outgrowth. Outgrowth was observed within 3 days. Neither attachment to substrate nor outgrowth was noted from encapsulated glomeruli or those with tubular or extraglomerular vascular fragments. Medium was changed twice weekly. Confluence of cells surrounding glomer uli was noted within 7-14 days. Cells were passaged once using trypsin/EDTA solution (Gibco) at room temperature. The cell su spension was then diluted with an equal volume of cold Way mouth's MB 752/1 with 20% decomplemented serum to stop enzy matic digestion. This mixture was filtered through a No. 500 sieve (pore size 25 pm) to remove glomerular fragments and centrifuged at 400 g for 15 min to pellet the cells. These were resuspended in culture medium and plated at one third preharvest surface density. Cultures have been maintained after successive passage periods up to 6 months. Electron Microscopy Cell cultures at least 3 weeks and two passages old were plated onto glass cover slips which were previously rinsed in 70% ethanol, flame dried, then placed in small Petri dishes (Corning Glass Works, Corning, N.Y.). After I week in culture, prewarmed 8% gluteraldehyde was added directly to the culture medium to yield a final
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
76
concentration of 3%glutaraldehyde. The cells were fixed for 30 min at 24°C, rinsed twice in Hanks’ balanced salt solution (HBSS; GIBCO), left for 15 min in 2% osmium tetroxide in HBSS at 24°C, rinsed with veronal buffer, left for 15 min in 3% uranyl acetate in veronal buffer, rinsed again with veronal buffer, then serially dehy drated through ethanol. After 2 min in 1:1 ethanol :propylene oxide, then 2 min in pure propylene oxide, the coverslip was left in 1:1 propylene oxide:Epon overnight. The coverslip was subsequently drained and covered with Epon-filled Beem capsules for polymeri zation. Immunofluorescence Cells were grown on glass coverslips as described above. After 2-7 days in culture, coverslips were fixed in cold acetone (4°C) for 15 min, washed in PBS for 5 min, incubated at room temperature for 45 min with specific primary antibody, rinsed in PBS for 15 min, then incubated for 30 min with fluorescein (FITC) labeled second anti body. After 15 min in PBS, coverslips were mounted on glass slides and examined for immunofluorescence. The following primary anti-sera and dilutions were utilized: (a) rabbit anti-human factor Vlll-related antigen (Dako, Santa Barbara, Calif.), 1/41; (b) goat anti-chicken fibronectin (Calbiochetn Biochemicals, San Diego, Calif.), 1/21; (c) rabbit anti-chicken gizzard a-actinin (Miles Scien tific, Naperville, 111.), 1/101; (d) rabbit anti-bovine uterus myosin (Miles Scientific), 1/101; (e) T3, a mouse monoclonal lgG t, antibody with affinity for chicken macrophages. T lymphocytes, and erythro cytes (kind gift of Dr. Albert Benedict, University of Hawaii. Honol ulu), 1/1, and (0 mouse IgM monoclonal antibody to chicken la antigen (kind gift of Dr. Chen Lo Chen, University of Alabama, Birmingham), undiluted, and mouse monoclonal anti-human desmin (Dakopatts. Denmark), 1/20. Second antibodies used were: (a) FITC-labeled goat anti-rabbit IgG (Cooperbiomedical, Malvern, Pa.), 1/31: (b) FITC-labeled rabbit anti-goat IgG (Calbiochem Bio chemicals), 1/41, and (c) FITC-labeled goat anti-mouse immuno globulins (IgG. A, M; Cooperbiomedical), 1/11. Phagocytosis Latex beads (1.09 urn in diameter) in 10% aqueous suspension (Sigma) were added to cultured cells grown on glass coverslips at a concentration of 30 ul/10 ml culture medium. One hour later, coverslips were washed extensively with PBS, fixed 20 min in cold acetone (4°C), then mounted on glass slides. Cells were examined by phase contrast microscopy for phagocytosis. In some experi ments we examined the effects of fresh or heat in activated serum and cytochalasin B (Sigma) at I pg/ml. In others, Covasphere FX particles (Covalent Technology Corporation, Ann Arbor, Mich.) were used. They gave similar results to standard latex beads. Growth Chicken mesangial cells, between 1 and 2 months old, were harvested from tissue culture flasks and centrifuged as for passage, then resuspended in standard medium (20% FCS) with the concen tration adjusted to 1.5 x 10J cells/ml. Aliquots of 200 pi cell suspen sion were distributed in 96-well tissue culture trays (Costar) yielding 3,000 cells/well. Assays for tritiated thymidine uptake by cells exposed to growth factors and controls were run in triplicate. Twenty-four hours after cell plating, standard medium was aspi rated and replaced by test or control medium. Tritiated thymidine uptake for 3. 4, 5, and 6 days in experimental medium was deter mined by pulsing all wells 24 h prior to harvesting with 2 pCi of
77
tritiated thymidine (I mCi/ml, 6.7 C'i/mol; ICN Radiochemicals, Irvine, Calif.). Cells were harvested by aspirating medium, washing once with PBS, and adding 200 pi of trypsin/EDTA solution at room tempera ture (Gibco). The cells were observed under a microscope until they detached and rounded up. They were then collected onto glass filters (Titertek, available through Flow Laboratories. McLean, Va.) and washed using a cell harvester (Skatron, Sterling, Va.). Tritiated thymidine uptake was measured in cpm using a Beckman LS 230 liquid scintillation counter (Beckman Instruments, Fullerton, Ca lif.). In some experiments trypsinized cells were counted manually to confirm that tritiated thymidine uptake correlated with the in crease in cell number. Angiotensin II (All) Binding and Induction o f Contraction Second to fourth passage mesangial cells and chick embryo fibroblasts (CEF, see below) were washed with PBS and suspended in standard calcium-containing medium. Cells were incubated at 25°C for 30 min with ,:5I-A1I (2,200 Ci/mmol, NEN Research Products, Boston, Mass.). After washing, the cells were counted and background subtracted. All studies were performed in duplicate with I04 cells/tube. '-5I All was used in final concentrations of 10 7, 10 s, and 10“' M. An excess of unlabeled All (Sigma Chemical Company) w’as used to demonstrate displacement of the l25I-AII. For contraction studies, mesangial cells were rested in calciumand magnesium-free HBSS with 10% FCS. After overnight incuba tion in this medium, cells were incubated in H BSS with calcium and magnesium with All added to the final concentration of I nAito I pjV/ [24). Contraction of cells was examined both on standard plastic support and on fibronectin-coated plates. Fields of cells were isolated and photographed prior to addition of All and photo graphs then taken at 2- to 5-min intervals thereafter. Contraction studies were conducted at room temperature. Fibroblasts In order to compare chicken mesangial cells to chicken fibro blasts, we examined three different types of chicken fibroblasts. The first of these was derived from mincing chicken wattle and then culturing in the same medium used for glomeruli. The second type of chicken fibroblast was obtained commercially from American Type Culture Collection (ATCC), Rockville, Md.,and the third was primary CEF, a gift from Dr. Tom Parsons. The latter were prepared by isolating 9-day-old chick embryos, removing internal organs, limbs and head, then mincing, dispersing cells in trypsin and plating in Dulbecco’s modified Eagle's medium with 10% tryptose phos phate broth. These three widely divergent types of chick fibroblasts were chosen to compare to the glomerular outgrowths in terms of characterizing our cells as mesangial rather than fibroblastic. Mesangial cells and chicken fibroblasts were evaluated for their ability to grow in dialyzed medium supplemented with amino acids containing D- or /.-valine as described from mammalian fibroblasts (25). Morphology and growth characteristics of cells were evaluated and tritiated thymidine incorporation by the cells was quantitated. As a known control, 3T3 murine fibroblasts were grown under similar conditions and tritiated thymidine incorporation likewise evaluated. Statistical Analysis Data are presented as the m ean±SEM . Data were analyzed by Student’s t tests.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Chicken Mesangial Cell Culture
78
Sadovnic/Brand-Elnaggar/Bolton
Fig. !. a A large mammalian type chicken glomerulus attached to a plastic dish. Outgrowth of mesangial cells is apparent 3 days after attachment, x 320. b Two small reptilian-type chicken glomeruli have abundant growth of stellate and fusiform cells surrounding them 2 weeks after attachment, x 320.
Results Outgrowth from cultured chicken glomeruli (fig. 1) was noted by the 3rd day. Passaged cells formed conflu ent multilayered cultures without evidence of epithelial or endothelial cells which grow as monolayers of poly gonal cells [26, 27]. Chicken mesangial cells in culture appear to be a homogeneous cell type with a stellate or
fusiform shape. They grow in interwoven bundles and do not exhibit contact inhibition. Multilayered cells were present by the end of a month with formation of nodular mounds. Few or no cells grew from encapsulated glomer uli, and most of these glomeruli failed to attach. Chicken mesangial cells were sensitive to mitomycin C (Sigma) at 10 pg/ml with virtually complete detach ment of cells from substrate after 24 h. Adherence and
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Fig. 2. Electron micrograph of a chicken mesangial cell. Dense cyto plasmic microfilaments are abund antly present (M). These microfila ments demonstrate condensation toward the periphery of the cell and form an interlacing/interlocking network within the cellular cyto plasm. Multilamellar residual bod ies (arrows) are characteristically observed in chicken mesangial cells, x 5,000.
Fig. 3. Chicken mesangial cells demonstrating intense fibrillar immunofluorescence staining for fibronectin. x 320.
morphology were unaffected 96 h after the addition of aminonudeoside of puromycin (Sigma) to a final con centration of 100 ug/ml. On electron microscopy, chicken mesangial cells grew in multilayers as elongated cells with oval nuclei. They demonstrated a well-devel oped system of microfilaments apparent throughout the cytoplasm (fig. 2). These were most prominent peripher ally as parallel structures lying just below the cell mem brane. These cells contain moderate amounts of rough endoplasmic reticulum and numerous lysosomes. In ad dition, multilamellar residual bodies suggesting phago cytic activity were observed in chicken mesangial cells. Immunofluorescent staining of chicken mesangial cells was negative for factor Vlll-related antigen, as op posed to the granular pattern evident along the vascula ture of chicken kidney control. An intense fibrillar pat tern of immunofluorescence was present for fibronectin (fig. 3), «-actinin, desmin. and myosin. Immunofluores cence with the monoclonal antibody T3, which labels chicken T cells and macrophages, and monoclonal anti body directed against chicken la antigen were negative. Chicken mesangial cells avidly phagocytized latex beads (fig. 4). After I h of incubation, essentially 100% of cells had phagocytosed some beads, and uptake was inhibited by cytochalasin B (Sigma) at 1ug/ml. Phagocy tosis occurred in standard and serum-free medium, and with both fresh and heated serum. Phagocytic mesangial cells did not demonstrate surface staining for la antigen. Growth curves obtained by plotting tritiated thymi dine uptake vs. the number of days in culture medium are shown in figure 5. Curves for Waymouth’s medium with
79
Fig. 4. Phase contrast micrograph of chicken mesangial cells demonstrating phagocytosis of l-|im latex beads. The mesangial cells are engorged with the uptaken beads and are clearly distin guishable from background which contains essentially no beads. Costaining for chicken la antigen on phagocytic cells was negative, x 320.
Fig. 5. Chicken mesangial cell growth curves obtained from 24-hour tritiated thymidine uptake versus days in medium (mean±SEM ).
20,10, 5, and 2.5% FCS do not overlap and show optimal resolution on days 5 and 6. CEF did not display any binding of l25I-AI I at any of the concentrations studied. Mesangial cells did demon strate binding of All at each concentration examined. The binding was specific as indicated by displacement of l25I-AII by excess cold All (fig. 6). All-induced contraction was evident by phase mi croscopy and consisted of a decrease in cell size, round ing up and retraction of cell extensions. Contracted cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Chicken Mesangial Cell Culture
Fig. 6. Angiotensin II-binding assay showing specific binding to chicken mesangial cells in culture. Increasing concentrations of l25I-labelled A ll was added to tubes containing 5 x l0 5 chicken mesangial cells in buffer (20 mMTRIS-HCl, 125 m M NaCI, 5 m,V MgCIi, 0.2% BSA) and ACTH 12 pg/ml. Nonspecific background has been subtracted and net counts are presented. Specific binding increased with increasing amounts of All and was specifically blocked by a 100-fold excessof non-labcled com petitor(■). Concen tration time points were performed in triplicate (mean±SEM ).
7b
Sadovnic/Brand-Elnaggar Bolton
had condensation of cytoplasm associated with the shape change. All-induced contraction of 25-40% of cells grown on uncoated and fibronectin-coated plastic Petri dishes (fig. 7). Chicken mesangial cells were clearly distinguishable from fibroblasts (fig. 8). Fibroblasts grew much more rapidly to confluence, within 3-5 days, while mesangial cells required 7 14 days. At confluence fibroblasts exhi bited contact inhibition and detached from substrate within a few days. On the other hand, mesangial cells continued to grow without detachment after reaching confluence and developed multilayered protuberances. By immunofluorescence fibroblasts did not stain for desmin. In D-valine substituted medium, mouse 3T3 cells dislodged and failed to grow (269±64 vs. 23,816±5,124 cpm). Chicken fibroblasts did not detach, but grew less
Fig. 7.a Phase contrast photograph of chicken mesangial cell grown overnight in calcium/magnesium-free IIBSS and 10% FC'S. Five mesangial cells are illustrated, b The same field of mesangial cells with medium containing calcium and magnesium 15 min after addition of I pMangiotensin II. Two of the five cells (arrows) have undergone contraction associated with retraction of cytoplasmic processes and rounding up of the cells with an increase in the refractile properties of the contracting cells, x 160. Fig. 8. Phase photo micrograph illustrating the morphology of chick embryo fibroblasts in primary culture 5 days after plating. Growth and morphologic differences from mesangial cells at 3 days and 2 weeks (fig. I) are apparent, x 100.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
80
Chicken Mesangial Cell Culture
well (1,035 ±405 vs. 1,484 ±405 cpm). No decreased growth was seen in mesangial cells in D-valine vs. /^va line (2,954 ±739 vs. 822 ±739 cpm). By electron micro scopy, fibroblasts lacked a well-developed microfilamentous system and did not contain the multilamellar bodies characteristic of mesangial cells. Furthermore, chicken fibroblasts did not phagocytose latex beads while mesangial cells were quite phagocytic. Finally, All did not bind to fibroblasts or induce contraction as ob served with mesangial cells.
Discussion Mesangial cells of mammalian origin have been cul tured from many different species since 1970 [27-33]. To our knowledge the isolation, growth, and characteriza tion of avian mesangial cells has not previously been described. Not unexpectably, methodology and condi tions utilized for mammalian mesangial cell culture were generally applicable to the successful culture of chicken
Table 1. Characteristics shared by cultured chicken and mam malian mesangial culture Characteristic
Mammalian Chicken mesangial cell mesangial cell
Overlapping stellate, strap-like.and spindle-shaped cells by light microscopy Well-developed microfilamentous system ultrastructurally Resistant to aminonucleoside of puromycin to 100 pg/ml Sensitive to mitomycin C at 10 gg/ml Grow in D-valine-containig L-valine-free medium Immunofluorescence (IF) for factor Vlll-related antigen Intense IF for fibronectin, desmin Fibrillar IF for u-actinin, myosin Phagocytize polystyrene beads
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
yes yes yes, when opsonized no
yes yes yes
yes, but substrate dependent, percentage variable
yes, but substrate dependent: 30%
IF for macrophage and common leukocyte antigens Bind A I1and contract on exposure to All
na
mesangial cells. It was, however, necessary to make some alterations in the preparation of kidneys for the growth of mesangial cells from chicken glomeruli because of the unique anatomic structure of the avian kidney. Utiliza tion of a harsher isolation procedure with homogeniza tion and collagenase digestion resulted in our ability to grow mesangial cells. This probably contributed to the lack of contamination with other cells which were more susceptible to these harsher isolation procedures. Condi tions required for maintenance of cells in culture were similar to those in mammalian mesangial cells, including the requirement for higher concentrations of serum for optimal growth and the use of either Waymouth's or RPMI. Chicken mesangial cells share many characteristics with cultured mammalian mesangial cells (table 1). Cells grow in a random pattern with strap-like morphology in overlaping bundles and appear to be a homogeneous cell type. Like mammalian mesangial cells, chicken mesan gial cells maintained in culture for prolonged periods of time continue to grow by heaping up upon each other, forming ridges and hills and small protuberances of mul tilayered cells. Even though some primary and secondary cultures were allowed to grow for 10 weeks with resultant formtion of multilayers, we did not detect large 'hillocks’ as described by Sterzel et al. [34]. The chicken mesangial cells did not demonstrate contact inhibition of growth nor did they detach from plates as the density of the cells increased as seen with fibroblasts. The mesangial cells contained a well-developed microfilamentous system with multiple microfibrils running parallel to the cell surface and forming interlacing networks within the cell by electron microscopy. Intense fibrillar staining was seen with antisera to a-actinin and myosin as well as fibronectin. In addition, mesangial cells in cultures stained intensely for desmin as described by Yaoita et al. [35]. No staining was seen for factor VIII on any of the cells indicating that they were not endothelial in origin. Factor VIII staining was not observed in glomeruli of chicken kidney sections although endothelial structures did stain. Contamination with large cobblestone poly gonal cells characteristic of epithelial cells was not seen and only once when growing primary cells in 2% FCS did we detect cells of probable epithelial origin (fig. 9). Like mammalian mesangial cells, chicken mesangial cells were resistant to aminonucleoside of puromycin and sensitive to mitomycin C. Chicken mesangial cells were unexpectably avidly phagocytic for polystyrene particles. Both latex and Covaspheres were taken up by mesangial cells in culture.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
81
Sadovnic/Brand-Elnaggar/Bolton
CEFdid not demonstrate any binding of AM inanyof the assays. Mesangial cells did bind l25I-AI I as described in rats and humans [39,40]. This binding was specific for All since excess unlabeled All displaced l25I-AII. Addi tion of All to chicken mesangial cells rested overnight in calcium/magnesium-free medium resulted in contrac tion of approximately 30% of cells observed by phase microscopy. Theses cells were grown on plastic or fibronectin. These percents are similar to those reported by others in mammalian mesangial cells in culture. Most investigators have reported approximately 20-40% con traction on plastic [39, 41-43]. Mesangial cells grown on substrates which allow less tight cell adherence demon Fig. 9. A colony of chicken renal epithelial cells surrounding a strate a higher percentage of contraction [32,44], Studies in cloned cells describe contraction of 100% of cells [24], reptilian type glomerulus. These very large flat polygonal cells grow in a monolayer. They were cultured using a FCS concentration of Thus, response to All in chicken mesangial cells appears 2.5% which does not favor mesangial cell growth, x 320. comparable to that of mammalian mesangial cells under similar conditions. Chicken mesangial cells in culture grew as a homo geneous cell type comparable to mesangial cells from mammalian species. Fibroblastic contamination in ro The degree of uptake of latex particles varied from cell to dents can be easily ascertained by substitution of D-\acell with some cells having only a half a dozen or so line for L-valine in culture medium. Rodent fibroblasts particles per cell while others were engorged. Uptake was lack D-amino acid oxidase [25], On exposure to £>-valinenot complement dependent since both fresh and heated substituted medium, rodent fibroblasts detach and fail to serum work equally well. Cells did not appear to be grow. The response of other mammalian fibroblasts is macrophages since so-staining of phagocytic cells for la less dramatic and detachment may not occur, however, antigen was negative. In addition, most cells took up some inhibition of cell growth despite lack of detachment some particles of latex. If there were contamination with is observed in human fibroblasts [25]. We examined the macrophages to account for the phagocytosis, these growth characteristics of chicken fibroblasts, chicken should be a small percentage of cells. Others have re mesangial cells, and 3T3 murine fibroblasts in D- and ported phagocytosis in mesangial cells. Baud et al. [36] L-valine-supplemented media. As previously described and Schlondorf et al. [37] described the requirement for for rodents, 3T3 cells detached and failed to grow in complement for optimal uptake of zymosan particles D-valine. Chicken mesangial cells grew equally well in Dalthough some phagocytosis was present even in the and L-valine-substituted medium. Chicken fibroblasts absence complement [38]. Uptake was time dependent, grown in D-valine did not detach from plates but did have with most mesangial cells demonstrating particles by 1 h some supression of growth. Although we did not stain for of incubation [38]. Complement was necessary for PGE2 D-amino acid oxidase, chicken fibroblasts may contain production. In rats the uptake of immune complexes or low levels of this enzyme and thus be less susceptible to serum-coated colloidal gold particles appears to be via £)-valine. Other characteristics of chicken mesangial cells coated pits with incorporation into vesicles and lyso- and fibroblasts, however, clearly distinguished these two somes and is complement dependent [38]. Phagocytosis types of cells. Mesangial cells grew for many weeks in chicken mesangial cells was completely inhibited by without passage resulting in multilayered heaped up cells cytochalasin B as described in rats [38]. The present which did not show contact inhibition and did not detach studies have not further investigated the kinetics or mech from plates. Chicken fibroblasts demonstrated contact anisms involved in the phagocytic event nor products inhibition and detached from plate 3-5 days after reach released during this process. Future studies will be re ing confluence. Secondly, the pattern of growth of quired to further characterize phagocytosis in chicken chicken fibroblasts was quite different from mesangial mesangial cells to compare this phenomenon to mam cells. Three to five days of growth resulted in confluence malian mesangial cells. of fibroblasts but nearly 2 weeks were required for mes-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
82
Chicken Mesangial Cell Culture
4
5 6
7
8
9
10
11
12
13
14
15
Acknowledgments The authors thank Ms. Joyce Henderson and Ms. Gwen Binz for their expert secretarial assistance in preparing the manuscript and Walter May for his technical assistance.
References 1 Bolton WK, Sturgill BC: Proliferative glomerulonephritis in cluding post-infectious, non-infectious and crescentic glomer ulonephritis: in TisherCC, Brenner BM (eds): Renal Pathology. Philadelphia. Lippincott. 1989, pp 156-195. 2 Bolton WK: The role of high dose steroids in nephrotic syn drome: The case for aggressive use; in Narins RG (ed): Con troversies in Nephrology and Hypertension. Edinburgh. Chur chill-Livingston, 1984. pp 421-459. 3 Stilmant MM. Bolton WK, Sturgill BC.et al: Crescenticglomer-
16
17
18
19
20
21
ulonephritis without immune deposits: Clinicopathologic fea tures. Kidney Int 1979:15:184-195. Bolton WK:The role of cell mediated immunity in the pathogen esis of glomerulonephritis. Plasma Ther Transfusion Tech 1984; 5:415-430. Fillit HM, Zabriskie JB: Cellular immunity in glomerulonephri tis. Am J Pathol 1982:109:227-243. Steblay RW: Glomerulonephritis induced in sheep by injections of heterologous glomerular basement membrane and Freund's complete adjuvant. J Exp Med 1962:116:253-271. Williams R, Steblay R: Glomerulonephritis induced in goats by injections of human glomerular basement membrane and Freund's adjuvant. Fed Proc 1965:24:243. Bolton WK. Benton FR, Sturgill BC: Autoimmune glomerulo tubular nephropathy in mice. Clin Exp Immunol 1978:33: 463-467. Moulonguet-Doleris D. Erard D. Auffredou MT. et al: Speci ficity of antibodies to heterologous glomerular and tubular basement membranes in various strains of mice with different H-2 types. Clin Exp Immunol 1981 ;46:35~43. Matsuo S, Brentjens JR, Andres G, et al: Distribution of base ment membrane antigens in glomeruli of mice with autoimmune glomerulonephritis. Am J Pathol 1986:122:36-49. Sado Y, Okigaki T, Takamiya H, et al: Experimental autoim mune glomerulonephritis with pulmonary hemorrhage in rats. The dose-effect relationship of the nephritogenic antigen from bovine glomerular basement membrane. Clin Lab Immunol 1984;15:199-204. Unanue ER, Dixon FJ: Experimental allergic glomerulonephri tis induced in the rabbit with heterologous renal antigens. J Exp Med 1967;125:149-162. Unanue ER, Dixon FJ, Feldman JD: Experimental allergic glomerulonephritis induced in the rabbit with homologous renal antigens. J Exp Med 1967:125:163-183. Couser WG, Stilmant M, Lewis EJ: Experimental glomerulone phritis in the guinea pig. I. Glomerular lesions associated with antiglomerular basement membrane antibody deposits. Lab in vest 1973:29:236-243. Couser WG, Spargo BH, Stilmant MM, et al: Experimental glomerulonephritis in the uinea pig. II. Ultrastructural lesions of the basement membrane associated with proteinuria. Lab Invest 1975:32:46-55. Steblay RW: Glomerulonephritis induced in monkeys by injec tions of heterologous glomerular basement membrane and Freund's adjuvant. Nature 1963:197:1173-1176. Bolton WK. Tucker FL, Sturgill BC: Experimental autoimmune glomerulonephritis in chickens. J Clin Lab Immunol 1980: 3:179-184. Bolton WK, Tucker FL, Sturgill BC: New avian model of experi mental glomerulonephritis consistent with mediation by cellular immunity. J Clin Invest 1984:73:1263-1276. Tucker FL, Sturgill BC, Bolton WK: Ultrastructural studies of experimental autoimmune glomerulonephritis in normal and bursectomized chickens. Lab Invest 1985;53:563-570. Bolton WK, Chandra M. Tyson TM, et al: Transfer of experi mental glomerulonephritis in chickens by mononuclear cells. Kidney lnt 1988:34:598-610. Bolton WK, Atuk NO, Sturgill BC: Nephrotoxic nephritis in rabbits. The role of the sympathetic nervous system. Am J Pathol 1978:90:689-698.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
angial cells to reach confluence. Mesangial cells stained positively for desmin while chicken fibroblasts were ne gative. By electron microscopy mesangial cells had a well-developed filamentous system and multilamellar re sidual bodies, both of which were absent in fibroblasts. Chicken mesangial cells demonstrated avid phagocytosis of polystyrene beads, bound AII and underwent contrac tion with All. Fibroblasts did not take up polystyrene beads and did not have All binding or contraction induced with All. Finally, chicken mesangial cells in culture stain with monoclonal antibody directed against chicken mesangial cell epitopes and chicken fibroblasts are negative with this monoclonal anti body [45], Mammalian mesangial cells in culture have provided us with a wealth of information relative to normal mesangial cell function. They are proving to be of use as an in vitro model to examine pathogenetic events involved in im mune and nonimmune damage to glomeruli. In the pres ent paper we have described, for the first time, the culture and characterization of chicken mesangial cells and de monstrated similarities and dissimilarities to mammalian mesangial cells. Successful culture of chicken mesangial cells should allow us to examine in vitro pathogenetic processes operative in vivo in our model of EAG in chickens. By utilization of this in vitro system, we should be able to learn more about the role of cell-mediated immunity in the pathogenesis of EAG in chickens. We should consequently learn more of the role of cell-medi ated immunity in the pathogenesis of human glomerulo nephritis.
83
84
37 Schlondorff DJ, Satriano A, Hagege J, et al: Effect of platelet activating factor and serum treated by mosan on prostaglandin E; synthesis, arachidonic acid release, and contraction of cul tured rat mesangial cells. J Clin Invest 1984:73:1227-1231. 38 Singhal PC, Ding G, DeCandido S, et al: Endocytosis by cul tured mesangial cells and associated changes in prostaglandin E: synthesis. Am J Physiol 1987:252: F627-F634. 39 Foidart J, Sraer J, Delarue F, et al: Evidence for mesangial glomerular receptors for angiotensin II linked to mesangial cell contractility. FEBS Lett 1980:121:333-339. 40 Doi TM. Striker LJ. Elliot SJ.et al: Insulinlike growth factor-1 is a progression factor for human mesangial cells. Am J Pathol 1989:134:395-404. 41 Mahieu PR, Foidart JB. DuBois CH. et al: Tissue culture of normal rat glomeruli: Contractile activity of the cultured mesan gial cells. Invest Cell Pathol 1980;3:121-128. 42 Tanaka T, Fuji wara Y, Orita A, et al : The functional characteris tics of cultured rat mesangial cell. Jpn Cire J 1984;48:1017-1029. 43 Simonson MS, Dunn MJ: Leukotriene C4 and D4 contract rat glomerular mesangial cells. Kidney Int 1986;30:524-531. 4 4 Singhal PC, Scharschmidt LA, Gibbons N, et al: Contraction and relaxation of cultured mesangial cells on a silicone rubber surface. Kidney Int 1986:30:862-873. 45 Sadovnic MJ, Bolton WK: Proof that mesangial cells in culture are truly of mesangial origin. Clin Res 1988:36:597.
Accepted: June 26,1990 W. Kline Bolton. MD Division of Nephrology Department of Internal Medicine Box 133 University of Virginia Health Sciences Center Charlottesville. VA 22908 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
22 Dantzler WH. Braun EJ: Comparative nephron function in reptiles, birds, and mammals. Am J Physiol I980;293: R197R2I3. 23 Kreisberg Jl. Karnovsky MJ: Glomerular cells in culture. Kid ney Ini 1983;23:439-447. 24 Ausiello DA, Kreisberg J I, Roy C, et al: Contraction of cultured rat glomerular cells of apparent mesangial origin after stimula tion with angiotensin II and arginine vasopressin. J Clin Invest 1980;65:745-760. 25 Gilbert SF, Migeon BR: O-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 1975 ; 5 :11-17. 26 Striker GE, Soderland C, Bowen-Pope DF, et al: Isolation, characterization and propagation in vitro of human glomerular endothelial cells. J Exp Med 1984;160:323-328. 27 Striker GE, Striker LJ: Biology of disease: Glomerular cell culture. Lab Invest 1985:53:122-131. 28 Quadracci LJ, StrikerGE: Growth and maintenance ofglomerular cells in vitro. Proc Soc Exp Biol Med 1970:135:947-950. 29 Kreisberg. JI. Hoover RL, Karnovsky MJ: Isolation and charac terization of rat glomerular epithelial cells in vitro. Kidney Int 1978;14:21-30. 30 Scheinman JI, Fish AJ, Brown DM, et al: Human glomerular smooth muscle (mesangial) cells in culture. Lab Invest 1976; 34:150-158. 31 Norgaard JO: Cellular outgrowth from isolated glomeruli: Ori gin and characterization. Lab Invest 1983:48:526 542. 32 Kreisberg JI. Venkatachalam M, Troyer D: Contractile proper ties of cultured glomerular mesangial cells. Am J Physiol 1985:249 : F457-F463. 33 Schlondorff D: The glomerular mesangial cell: An expanding role for a specialized pericyte. FASEBJ 1987:1:272-281. 34 Sterzel RB. Lovett DH, Foellmer HG, et al: Mesangial cell Hillocks: Nodular foci of exaggerated growth of cells and matrix in prolonged culture. Am .1 Pathol 1986:125:130-140. 35 Yaoita E, Kazama T, Kawasaki K, et al: In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cell and dermal fibroblasts. Cell Pathol 1985:49:285 294. 36 Baud L, Hagege J, Sraer J, et al : Reactive oxygen production by cultured rat glomerular mesangial cells during phagocytosis is associated with stimulation of lipoxygenase activity. J Exp Med 1983:158:1836-1852.
Sadovnic/ Brand-Elnaggar/Bolton
Nephron 1991;58:75-84
Isolation and Characterization of Chicken Mesangial Cells1 Marc J. Sadovnica, Jessica Brand-Elnaggarb, W. Kline Boltonb a Division of Nephrology. Alleghany General Hospital, Pittsburgh, Pa, and l>Division of Nephrology, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Va., USA
Key Words. Mesangial cells • Cell culture, chicken • Non-mammalian, cell culture
Introduction Glomerulonephritis in man is associated with prolif eration of endogenous glomerular cells and an influx of exogenous inflammatory cells [1. 2]. The resultant histo logic lesions consist of variable degrees of glomerular tuft hypercellularity, at times associated with extracapillary proliferation. Anti-glomerular basement membrane (GBM) disease comprises 5% of all biopsies in patients with glomerulonephritis; however, approximately 25-30% of patients with a more malignant form of glo merulonephritis associated with rapidly progressive re nal failure (RPGN) have anti-GBM disease [1, 2]. An 1 This work was supported by USPHS grant DK 32530 from the NID D K .and NRSAgrant numbersT32 AM 07156-11 and5T32 HL 07284-10.
additional large subsegment of idiopathic RPGN con sists of those patients with minimal or no immune depos its, but with identical lesions by light microscopy [3]. Much evidence suggests a role for cell-mediated immu nity in many experimental models and in numerous types of glomerulonephritis in man [4, 5]. Unfortunately, few species lend themselves to the study of the role of cellmediated immunity in the pathogenesis of autoimmune glomerulonephritis. The model which most closely ap proximates anti-GBM human disease, Steblay nephritis, is induced by immunization of animals with heterologous GBM antigen [6], However, of mammalian species, only sheep and goats develop significant proliferative glomer ulonephritis, at times with crescents [6,7], GBM immuni zation of mice, rats, rabbits, guinea pigs, and monkeys results in either no detectable proliferation within glom eruli or variable proliferative changes (monkeys) [8-16].
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Abstract. Experimental autoimmune glomerulonephritis (EAG) in chickens appears to be mediated by cellular immunity and is associated with mesangial proliferation. We have developed techniques for the culture of chicken mesangial cells to study factors in vitro which lead to this proliferation. Chicken glomeruli isolated by sieving collagenase-treated whole kidney homogenates were cultured in Waymouth’s medium MB 752/1 supplemented with 20%decomplemented fetal calf serum and 1unit/ml insulin. Propagated cells share the following characteristics with mammalian mesangial cells: stellate and spindle-shaped morphology with an extensive microfilamentous system by light and electron microscopy; resistance to aminonucleoside of puromvein: susceptibility to mitomycin C : growth in L-valine-free medium; absent staining for factor Vlll-related antigen, chicken Tcell and la antigen: positive staining for fibronectin, myosin, a-actinin and desmin, and angiotensin II binding and induction of contraction. Unlike cultured mammalian mesangial cells, chicken mesangial cells avidly phagocytize latex beads and display multilamelIar residual bodies on electron microscopy indicative of phagocytic activity. They differed from fibroblasts which were non-phagocytic, had different growth patterns, fluorescence staining and ultrastructural morphology. To our knowledge, this is the first description of the culture of chicken mesangial cells. This in vitro system should allow further studies of pathogenetic processes involved in the production of EAG with elucidation of mechanisms relevant to human disease.
Sadov n ie /Brand-El nuggar/Bolton
Thus, despite immunization with GBM antigen and the development of antibodies to basement membrane, most mammalian species do not develop a proliferative glo merulonephritis. It is therefore difficult to examine the role of cell-mediated immunity in the pathogenesis of autoimmune glomerulonephritis in the absence of a pro liferative model in mammals. For these reasons we ex plored the development of autoimmune glomerulone phritis in chickens. We have reported that experimental autoimmune glomerulonephritis (EAG) can be induced in chickens by immunization with GBM antigen [17]. Normal chickens and birds made humorally incompetent through chemical bursectomy, then immunized with glomeruli, develop proliferative glomerulonephritis [18, 19]. Crescents are present in 3-28% of glomeruli depend ing on the strain and type of antigen utilized for immuni zation. EAG appears unrelated to the presence or ab sence of antibody bound in glomeruli and may be trans ferred to other chickens by mononuclear cells, but not by antibody [20]. Proliferation of mesangial cells is a major component of the hypercellularity in the glomerulone phritis in this model [19, 20], Since cell-mediated immu nity appears to be involved in the development of this mesangial hypercellularity in vivo, it would seem appro priate to examine the effect of various components of the cellular system in vitro with chicken mesangial cells. Although an extensive literature is available describing culture of mammalian mesangial cells, there is no infor mation available about culturing chicken mesangial cells. The purpose of the present report is to describe the methodology and techniques that we have developed to culture chicken mesangial cells and to characterize these cells in culture. Methods Glomerular Isolation Preparations of mammalian glomeruli predominantly free of tubular contamination and capsules may be obtained by passing cortical tissue through a series of graded metal sieves [21]. Several factors complicate the isolation of chicken glomeruli. The avian kidney lacks a well-demarcated cortex separating glomeruli from other renal tissue [20, 22]. Glomeruli are widely distributed in size with diameters ranging from 5 to 20 um (reptilian vs. mammalian), and small glomeruli are comparable in size to proximal tubules. Sieving of chicken kidney results in glomerular preparations heavily contaminated by tubules. In addition, this process fails to strip Bowman's capsule. Unencapsulated glomeruli are required for out growth in the chicken, as in other species [23]. To circumvent these difficulties, we explored a variety of techniques. The following procedure results in a preparation of unencapsulated glomeruli more than 80% free of tubular contamination.
Both kidneys are harvested aseptically from a single chicken, stripped of capsule and surrounding non-parenchymal tissue with forceps, then placed in a small tissue blender(Eberbach, Ann Arbor. Mich.) with ice-cold phosphate-buffered saline (PBS) to a final volume of 20 ml. Homogenization is carried out in six 15-second bursts separated by 15-second intervals to prevent tissue from over heating. The resultant mixture is added to a solution of 80 mg type-1 collagenase (Sigma. St. Louis, Mo.) in 50 ml of PBS which has been sterilized by passage through a 0,2-gm filter. Digestion is allowed to proceed at room temperature for 15 min with vigorous stirring. The resultant slurry is poured on top of four stacked 3-inch metal sieves (Newark Wire Cloth Co., Newark, N.J.) of mesh (upper to lower) No. 25. No. 140, No. 200, and No. 400 (opening size 710,107.74, and 38 gm:. respectively) resting on a beaker to catch the filtrate. Tissue is pressed through the upper sieves using a 25-ml Erlenmeyer flask as a pestle and washing with cold PBS forced from a syringe. Tissue caught on the No. 400 mesh is washed off with 30-40 ml of ice-cold PBS into a beaker and centrifuged at 400 g for 10 min. The supernat ant is discarded. Pellets are resuspended in culture medium and pooled. The final preparation consists primarily of unencapsulated glomeruli. Glomerular purity was 85% as previously described [20]. Resuspended glomeruli are distributed among 75-cmJ culture flasks (Costar. Cambridge, Mass.) with 25 ml of medium and placed in a humidified incubator with 5% CO» at 30°C. Glomerular Culture Multiple sera (fetal calf, chicken, goat, lamb) and culture media (Waymouth's MB 752/1, Dulbecco's modified Eagle's medium, minimum essential medium [MEM], RPMI 1640) were investigated. Outgrowth was most rapid with Waymouth’s medium MB 752/1 supplemented with 20% decomplemented fetal calf serum (FC'S), I unit/ml insulin, and antibiotics (standard medium): penicillin 100 units/ml, streptomycin 100 ug/ml, and amphotericin B 0.25 pg/ml (all media components :Gibco. Grand Island. N.Y.). Outgrowth and proliferation were favored by higher serum concentration. FCS proved better than goat serum and both were superior to lamb and chicken sera. Waymouth's medium provided a slight advantage over RPMI. Dulbecco's modified Eagle’s medium, and MEM resulted in poor mesangial cell outgrowth. Outgrowth was observed within 3 days. Neither attachment to substrate nor outgrowth was noted from encapsulated glomeruli or those with tubular or extraglomerular vascular fragments. Medium was changed twice weekly. Confluence of cells surrounding glomer uli was noted within 7-14 days. Cells were passaged once using trypsin/EDTA solution (Gibco) at room temperature. The cell su spension was then diluted with an equal volume of cold Way mouth's MB 752/1 with 20% decomplemented serum to stop enzy matic digestion. This mixture was filtered through a No. 500 sieve (pore size 25 pm) to remove glomerular fragments and centrifuged at 400 g for 15 min to pellet the cells. These were resuspended in culture medium and plated at one third preharvest surface density. Cultures have been maintained after successive passage periods up to 6 months. Electron Microscopy Cell cultures at least 3 weeks and two passages old were plated onto glass cover slips which were previously rinsed in 70% ethanol, flame dried, then placed in small Petri dishes (Corning Glass Works, Corning, N.Y.). After I week in culture, prewarmed 8% gluteraldehyde was added directly to the culture medium to yield a final
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
76
concentration of 3%glutaraldehyde. The cells were fixed for 30 min at 24°C, rinsed twice in Hanks’ balanced salt solution (HBSS; GIBCO), left for 15 min in 2% osmium tetroxide in HBSS at 24°C, rinsed with veronal buffer, left for 15 min in 3% uranyl acetate in veronal buffer, rinsed again with veronal buffer, then serially dehy drated through ethanol. After 2 min in 1:1 ethanol :propylene oxide, then 2 min in pure propylene oxide, the coverslip was left in 1:1 propylene oxide:Epon overnight. The coverslip was subsequently drained and covered with Epon-filled Beem capsules for polymeri zation. Immunofluorescence Cells were grown on glass coverslips as described above. After 2-7 days in culture, coverslips were fixed in cold acetone (4°C) for 15 min, washed in PBS for 5 min, incubated at room temperature for 45 min with specific primary antibody, rinsed in PBS for 15 min, then incubated for 30 min with fluorescein (FITC) labeled second anti body. After 15 min in PBS, coverslips were mounted on glass slides and examined for immunofluorescence. The following primary anti-sera and dilutions were utilized: (a) rabbit anti-human factor Vlll-related antigen (Dako, Santa Barbara, Calif.), 1/41; (b) goat anti-chicken fibronectin (Calbiochetn Biochemicals, San Diego, Calif.), 1/21; (c) rabbit anti-chicken gizzard a-actinin (Miles Scien tific, Naperville, 111.), 1/101; (d) rabbit anti-bovine uterus myosin (Miles Scientific), 1/101; (e) T3, a mouse monoclonal lgG t, antibody with affinity for chicken macrophages. T lymphocytes, and erythro cytes (kind gift of Dr. Albert Benedict, University of Hawaii. Honol ulu), 1/1, and (0 mouse IgM monoclonal antibody to chicken la antigen (kind gift of Dr. Chen Lo Chen, University of Alabama, Birmingham), undiluted, and mouse monoclonal anti-human desmin (Dakopatts. Denmark), 1/20. Second antibodies used were: (a) FITC-labeled goat anti-rabbit IgG (Cooperbiomedical, Malvern, Pa.), 1/31: (b) FITC-labeled rabbit anti-goat IgG (Calbiochem Bio chemicals), 1/41, and (c) FITC-labeled goat anti-mouse immuno globulins (IgG. A, M; Cooperbiomedical), 1/11. Phagocytosis Latex beads (1.09 urn in diameter) in 10% aqueous suspension (Sigma) were added to cultured cells grown on glass coverslips at a concentration of 30 ul/10 ml culture medium. One hour later, coverslips were washed extensively with PBS, fixed 20 min in cold acetone (4°C), then mounted on glass slides. Cells were examined by phase contrast microscopy for phagocytosis. In some experi ments we examined the effects of fresh or heat in activated serum and cytochalasin B (Sigma) at I pg/ml. In others, Covasphere FX particles (Covalent Technology Corporation, Ann Arbor, Mich.) were used. They gave similar results to standard latex beads. Growth Chicken mesangial cells, between 1 and 2 months old, were harvested from tissue culture flasks and centrifuged as for passage, then resuspended in standard medium (20% FCS) with the concen tration adjusted to 1.5 x 10J cells/ml. Aliquots of 200 pi cell suspen sion were distributed in 96-well tissue culture trays (Costar) yielding 3,000 cells/well. Assays for tritiated thymidine uptake by cells exposed to growth factors and controls were run in triplicate. Twenty-four hours after cell plating, standard medium was aspi rated and replaced by test or control medium. Tritiated thymidine uptake for 3. 4, 5, and 6 days in experimental medium was deter mined by pulsing all wells 24 h prior to harvesting with 2 pCi of
77
tritiated thymidine (I mCi/ml, 6.7 C'i/mol; ICN Radiochemicals, Irvine, Calif.). Cells were harvested by aspirating medium, washing once with PBS, and adding 200 pi of trypsin/EDTA solution at room tempera ture (Gibco). The cells were observed under a microscope until they detached and rounded up. They were then collected onto glass filters (Titertek, available through Flow Laboratories. McLean, Va.) and washed using a cell harvester (Skatron, Sterling, Va.). Tritiated thymidine uptake was measured in cpm using a Beckman LS 230 liquid scintillation counter (Beckman Instruments, Fullerton, Ca lif.). In some experiments trypsinized cells were counted manually to confirm that tritiated thymidine uptake correlated with the in crease in cell number. Angiotensin II (All) Binding and Induction o f Contraction Second to fourth passage mesangial cells and chick embryo fibroblasts (CEF, see below) were washed with PBS and suspended in standard calcium-containing medium. Cells were incubated at 25°C for 30 min with ,:5I-A1I (2,200 Ci/mmol, NEN Research Products, Boston, Mass.). After washing, the cells were counted and background subtracted. All studies were performed in duplicate with I04 cells/tube. '-5I All was used in final concentrations of 10 7, 10 s, and 10“' M. An excess of unlabeled All (Sigma Chemical Company) w’as used to demonstrate displacement of the l25I-AII. For contraction studies, mesangial cells were rested in calciumand magnesium-free HBSS with 10% FCS. After overnight incuba tion in this medium, cells were incubated in H BSS with calcium and magnesium with All added to the final concentration of I nAito I pjV/ [24). Contraction of cells was examined both on standard plastic support and on fibronectin-coated plates. Fields of cells were isolated and photographed prior to addition of All and photo graphs then taken at 2- to 5-min intervals thereafter. Contraction studies were conducted at room temperature. Fibroblasts In order to compare chicken mesangial cells to chicken fibro blasts, we examined three different types of chicken fibroblasts. The first of these was derived from mincing chicken wattle and then culturing in the same medium used for glomeruli. The second type of chicken fibroblast was obtained commercially from American Type Culture Collection (ATCC), Rockville, Md.,and the third was primary CEF, a gift from Dr. Tom Parsons. The latter were prepared by isolating 9-day-old chick embryos, removing internal organs, limbs and head, then mincing, dispersing cells in trypsin and plating in Dulbecco’s modified Eagle's medium with 10% tryptose phos phate broth. These three widely divergent types of chick fibroblasts were chosen to compare to the glomerular outgrowths in terms of characterizing our cells as mesangial rather than fibroblastic. Mesangial cells and chicken fibroblasts were evaluated for their ability to grow in dialyzed medium supplemented with amino acids containing D- or /.-valine as described from mammalian fibroblasts (25). Morphology and growth characteristics of cells were evaluated and tritiated thymidine incorporation by the cells was quantitated. As a known control, 3T3 murine fibroblasts were grown under similar conditions and tritiated thymidine incorporation likewise evaluated. Statistical Analysis Data are presented as the m ean±SEM . Data were analyzed by Student’s t tests.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Chicken Mesangial Cell Culture
78
Sadovnic/Brand-Elnaggar/Bolton
Fig. !. a A large mammalian type chicken glomerulus attached to a plastic dish. Outgrowth of mesangial cells is apparent 3 days after attachment, x 320. b Two small reptilian-type chicken glomeruli have abundant growth of stellate and fusiform cells surrounding them 2 weeks after attachment, x 320.
Results Outgrowth from cultured chicken glomeruli (fig. 1) was noted by the 3rd day. Passaged cells formed conflu ent multilayered cultures without evidence of epithelial or endothelial cells which grow as monolayers of poly gonal cells [26, 27]. Chicken mesangial cells in culture appear to be a homogeneous cell type with a stellate or
fusiform shape. They grow in interwoven bundles and do not exhibit contact inhibition. Multilayered cells were present by the end of a month with formation of nodular mounds. Few or no cells grew from encapsulated glomer uli, and most of these glomeruli failed to attach. Chicken mesangial cells were sensitive to mitomycin C (Sigma) at 10 pg/ml with virtually complete detach ment of cells from substrate after 24 h. Adherence and
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Fig. 2. Electron micrograph of a chicken mesangial cell. Dense cyto plasmic microfilaments are abund antly present (M). These microfila ments demonstrate condensation toward the periphery of the cell and form an interlacing/interlocking network within the cellular cyto plasm. Multilamellar residual bod ies (arrows) are characteristically observed in chicken mesangial cells, x 5,000.
Fig. 3. Chicken mesangial cells demonstrating intense fibrillar immunofluorescence staining for fibronectin. x 320.
morphology were unaffected 96 h after the addition of aminonudeoside of puromycin (Sigma) to a final con centration of 100 ug/ml. On electron microscopy, chicken mesangial cells grew in multilayers as elongated cells with oval nuclei. They demonstrated a well-devel oped system of microfilaments apparent throughout the cytoplasm (fig. 2). These were most prominent peripher ally as parallel structures lying just below the cell mem brane. These cells contain moderate amounts of rough endoplasmic reticulum and numerous lysosomes. In ad dition, multilamellar residual bodies suggesting phago cytic activity were observed in chicken mesangial cells. Immunofluorescent staining of chicken mesangial cells was negative for factor Vlll-related antigen, as op posed to the granular pattern evident along the vascula ture of chicken kidney control. An intense fibrillar pat tern of immunofluorescence was present for fibronectin (fig. 3), «-actinin, desmin. and myosin. Immunofluores cence with the monoclonal antibody T3, which labels chicken T cells and macrophages, and monoclonal anti body directed against chicken la antigen were negative. Chicken mesangial cells avidly phagocytized latex beads (fig. 4). After I h of incubation, essentially 100% of cells had phagocytosed some beads, and uptake was inhibited by cytochalasin B (Sigma) at 1ug/ml. Phagocy tosis occurred in standard and serum-free medium, and with both fresh and heated serum. Phagocytic mesangial cells did not demonstrate surface staining for la antigen. Growth curves obtained by plotting tritiated thymi dine uptake vs. the number of days in culture medium are shown in figure 5. Curves for Waymouth’s medium with
79
Fig. 4. Phase contrast micrograph of chicken mesangial cells demonstrating phagocytosis of l-|im latex beads. The mesangial cells are engorged with the uptaken beads and are clearly distin guishable from background which contains essentially no beads. Costaining for chicken la antigen on phagocytic cells was negative, x 320.
Fig. 5. Chicken mesangial cell growth curves obtained from 24-hour tritiated thymidine uptake versus days in medium (mean±SEM ).
20,10, 5, and 2.5% FCS do not overlap and show optimal resolution on days 5 and 6. CEF did not display any binding of l25I-AI I at any of the concentrations studied. Mesangial cells did demon strate binding of All at each concentration examined. The binding was specific as indicated by displacement of l25I-AII by excess cold All (fig. 6). All-induced contraction was evident by phase mi croscopy and consisted of a decrease in cell size, round ing up and retraction of cell extensions. Contracted cells
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
Chicken Mesangial Cell Culture
Fig. 6. Angiotensin II-binding assay showing specific binding to chicken mesangial cells in culture. Increasing concentrations of l25I-labelled A ll was added to tubes containing 5 x l0 5 chicken mesangial cells in buffer (20 mMTRIS-HCl, 125 m M NaCI, 5 m,V MgCIi, 0.2% BSA) and ACTH 12 pg/ml. Nonspecific background has been subtracted and net counts are presented. Specific binding increased with increasing amounts of All and was specifically blocked by a 100-fold excessof non-labcled com petitor(■). Concen tration time points were performed in triplicate (mean±SEM ).
7b
Sadovnic/Brand-Elnaggar Bolton
had condensation of cytoplasm associated with the shape change. All-induced contraction of 25-40% of cells grown on uncoated and fibronectin-coated plastic Petri dishes (fig. 7). Chicken mesangial cells were clearly distinguishable from fibroblasts (fig. 8). Fibroblasts grew much more rapidly to confluence, within 3-5 days, while mesangial cells required 7 14 days. At confluence fibroblasts exhi bited contact inhibition and detached from substrate within a few days. On the other hand, mesangial cells continued to grow without detachment after reaching confluence and developed multilayered protuberances. By immunofluorescence fibroblasts did not stain for desmin. In D-valine substituted medium, mouse 3T3 cells dislodged and failed to grow (269±64 vs. 23,816±5,124 cpm). Chicken fibroblasts did not detach, but grew less
Fig. 7.a Phase contrast photograph of chicken mesangial cell grown overnight in calcium/magnesium-free IIBSS and 10% FC'S. Five mesangial cells are illustrated, b The same field of mesangial cells with medium containing calcium and magnesium 15 min after addition of I pMangiotensin II. Two of the five cells (arrows) have undergone contraction associated with retraction of cytoplasmic processes and rounding up of the cells with an increase in the refractile properties of the contracting cells, x 160. Fig. 8. Phase photo micrograph illustrating the morphology of chick embryo fibroblasts in primary culture 5 days after plating. Growth and morphologic differences from mesangial cells at 3 days and 2 weeks (fig. I) are apparent, x 100.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
80
Chicken Mesangial Cell Culture
well (1,035 ±405 vs. 1,484 ±405 cpm). No decreased growth was seen in mesangial cells in D-valine vs. /^va line (2,954 ±739 vs. 822 ±739 cpm). By electron micro scopy, fibroblasts lacked a well-developed microfilamentous system and did not contain the multilamellar bodies characteristic of mesangial cells. Furthermore, chicken fibroblasts did not phagocytose latex beads while mesangial cells were quite phagocytic. Finally, All did not bind to fibroblasts or induce contraction as ob served with mesangial cells.
Discussion Mesangial cells of mammalian origin have been cul tured from many different species since 1970 [27-33]. To our knowledge the isolation, growth, and characteriza tion of avian mesangial cells has not previously been described. Not unexpectably, methodology and condi tions utilized for mammalian mesangial cell culture were generally applicable to the successful culture of chicken
Table 1. Characteristics shared by cultured chicken and mam malian mesangial culture Characteristic
Mammalian Chicken mesangial cell mesangial cell
Overlapping stellate, strap-like.and spindle-shaped cells by light microscopy Well-developed microfilamentous system ultrastructurally Resistant to aminonucleoside of puromycin to 100 pg/ml Sensitive to mitomycin C at 10 gg/ml Grow in D-valine-containig L-valine-free medium Immunofluorescence (IF) for factor Vlll-related antigen Intense IF for fibronectin, desmin Fibrillar IF for u-actinin, myosin Phagocytize polystyrene beads
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
yes yes yes, when opsonized no
yes yes yes
yes, but substrate dependent, percentage variable
yes, but substrate dependent: 30%
IF for macrophage and common leukocyte antigens Bind A I1and contract on exposure to All
na
mesangial cells. It was, however, necessary to make some alterations in the preparation of kidneys for the growth of mesangial cells from chicken glomeruli because of the unique anatomic structure of the avian kidney. Utiliza tion of a harsher isolation procedure with homogeniza tion and collagenase digestion resulted in our ability to grow mesangial cells. This probably contributed to the lack of contamination with other cells which were more susceptible to these harsher isolation procedures. Condi tions required for maintenance of cells in culture were similar to those in mammalian mesangial cells, including the requirement for higher concentrations of serum for optimal growth and the use of either Waymouth's or RPMI. Chicken mesangial cells share many characteristics with cultured mammalian mesangial cells (table 1). Cells grow in a random pattern with strap-like morphology in overlaping bundles and appear to be a homogeneous cell type. Like mammalian mesangial cells, chicken mesan gial cells maintained in culture for prolonged periods of time continue to grow by heaping up upon each other, forming ridges and hills and small protuberances of mul tilayered cells. Even though some primary and secondary cultures were allowed to grow for 10 weeks with resultant formtion of multilayers, we did not detect large 'hillocks’ as described by Sterzel et al. [34]. The chicken mesangial cells did not demonstrate contact inhibition of growth nor did they detach from plates as the density of the cells increased as seen with fibroblasts. The mesangial cells contained a well-developed microfilamentous system with multiple microfibrils running parallel to the cell surface and forming interlacing networks within the cell by electron microscopy. Intense fibrillar staining was seen with antisera to a-actinin and myosin as well as fibronectin. In addition, mesangial cells in cultures stained intensely for desmin as described by Yaoita et al. [35]. No staining was seen for factor VIII on any of the cells indicating that they were not endothelial in origin. Factor VIII staining was not observed in glomeruli of chicken kidney sections although endothelial structures did stain. Contamination with large cobblestone poly gonal cells characteristic of epithelial cells was not seen and only once when growing primary cells in 2% FCS did we detect cells of probable epithelial origin (fig. 9). Like mammalian mesangial cells, chicken mesangial cells were resistant to aminonucleoside of puromycin and sensitive to mitomycin C. Chicken mesangial cells were unexpectably avidly phagocytic for polystyrene particles. Both latex and Covaspheres were taken up by mesangial cells in culture.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
81
Sadovnic/Brand-Elnaggar/Bolton
CEFdid not demonstrate any binding of AM inanyof the assays. Mesangial cells did bind l25I-AI I as described in rats and humans [39,40]. This binding was specific for All since excess unlabeled All displaced l25I-AII. Addi tion of All to chicken mesangial cells rested overnight in calcium/magnesium-free medium resulted in contrac tion of approximately 30% of cells observed by phase microscopy. Theses cells were grown on plastic or fibronectin. These percents are similar to those reported by others in mammalian mesangial cells in culture. Most investigators have reported approximately 20-40% con traction on plastic [39, 41-43]. Mesangial cells grown on substrates which allow less tight cell adherence demon Fig. 9. A colony of chicken renal epithelial cells surrounding a strate a higher percentage of contraction [32,44], Studies in cloned cells describe contraction of 100% of cells [24], reptilian type glomerulus. These very large flat polygonal cells grow in a monolayer. They were cultured using a FCS concentration of Thus, response to All in chicken mesangial cells appears 2.5% which does not favor mesangial cell growth, x 320. comparable to that of mammalian mesangial cells under similar conditions. Chicken mesangial cells in culture grew as a homo geneous cell type comparable to mesangial cells from mammalian species. Fibroblastic contamination in ro The degree of uptake of latex particles varied from cell to dents can be easily ascertained by substitution of D-\acell with some cells having only a half a dozen or so line for L-valine in culture medium. Rodent fibroblasts particles per cell while others were engorged. Uptake was lack D-amino acid oxidase [25], On exposure to £>-valinenot complement dependent since both fresh and heated substituted medium, rodent fibroblasts detach and fail to serum work equally well. Cells did not appear to be grow. The response of other mammalian fibroblasts is macrophages since so-staining of phagocytic cells for la less dramatic and detachment may not occur, however, antigen was negative. In addition, most cells took up some inhibition of cell growth despite lack of detachment some particles of latex. If there were contamination with is observed in human fibroblasts [25]. We examined the macrophages to account for the phagocytosis, these growth characteristics of chicken fibroblasts, chicken should be a small percentage of cells. Others have re mesangial cells, and 3T3 murine fibroblasts in D- and ported phagocytosis in mesangial cells. Baud et al. [36] L-valine-supplemented media. As previously described and Schlondorf et al. [37] described the requirement for for rodents, 3T3 cells detached and failed to grow in complement for optimal uptake of zymosan particles D-valine. Chicken mesangial cells grew equally well in Dalthough some phagocytosis was present even in the and L-valine-substituted medium. Chicken fibroblasts absence complement [38]. Uptake was time dependent, grown in D-valine did not detach from plates but did have with most mesangial cells demonstrating particles by 1 h some supression of growth. Although we did not stain for of incubation [38]. Complement was necessary for PGE2 D-amino acid oxidase, chicken fibroblasts may contain production. In rats the uptake of immune complexes or low levels of this enzyme and thus be less susceptible to serum-coated colloidal gold particles appears to be via £)-valine. Other characteristics of chicken mesangial cells coated pits with incorporation into vesicles and lyso- and fibroblasts, however, clearly distinguished these two somes and is complement dependent [38]. Phagocytosis types of cells. Mesangial cells grew for many weeks in chicken mesangial cells was completely inhibited by without passage resulting in multilayered heaped up cells cytochalasin B as described in rats [38]. The present which did not show contact inhibition and did not detach studies have not further investigated the kinetics or mech from plates. Chicken fibroblasts demonstrated contact anisms involved in the phagocytic event nor products inhibition and detached from plate 3-5 days after reach released during this process. Future studies will be re ing confluence. Secondly, the pattern of growth of quired to further characterize phagocytosis in chicken chicken fibroblasts was quite different from mesangial mesangial cells to compare this phenomenon to mam cells. Three to five days of growth resulted in confluence malian mesangial cells. of fibroblasts but nearly 2 weeks were required for mes-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
82
Chicken Mesangial Cell Culture
4
5 6
7
8
9
10
11
12
13
14
15
Acknowledgments The authors thank Ms. Joyce Henderson and Ms. Gwen Binz for their expert secretarial assistance in preparing the manuscript and Walter May for his technical assistance.
References 1 Bolton WK, Sturgill BC: Proliferative glomerulonephritis in cluding post-infectious, non-infectious and crescentic glomer ulonephritis: in TisherCC, Brenner BM (eds): Renal Pathology. Philadelphia. Lippincott. 1989, pp 156-195. 2 Bolton WK: The role of high dose steroids in nephrotic syn drome: The case for aggressive use; in Narins RG (ed): Con troversies in Nephrology and Hypertension. Edinburgh. Chur chill-Livingston, 1984. pp 421-459. 3 Stilmant MM. Bolton WK, Sturgill BC.et al: Crescenticglomer-
16
17
18
19
20
21
ulonephritis without immune deposits: Clinicopathologic fea tures. Kidney Int 1979:15:184-195. Bolton WK:The role of cell mediated immunity in the pathogen esis of glomerulonephritis. Plasma Ther Transfusion Tech 1984; 5:415-430. Fillit HM, Zabriskie JB: Cellular immunity in glomerulonephri tis. Am J Pathol 1982:109:227-243. Steblay RW: Glomerulonephritis induced in sheep by injections of heterologous glomerular basement membrane and Freund's complete adjuvant. J Exp Med 1962:116:253-271. Williams R, Steblay R: Glomerulonephritis induced in goats by injections of human glomerular basement membrane and Freund's adjuvant. Fed Proc 1965:24:243. Bolton WK. Benton FR, Sturgill BC: Autoimmune glomerulo tubular nephropathy in mice. Clin Exp Immunol 1978:33: 463-467. Moulonguet-Doleris D. Erard D. Auffredou MT. et al: Speci ficity of antibodies to heterologous glomerular and tubular basement membranes in various strains of mice with different H-2 types. Clin Exp Immunol 1981 ;46:35~43. Matsuo S, Brentjens JR, Andres G, et al: Distribution of base ment membrane antigens in glomeruli of mice with autoimmune glomerulonephritis. Am J Pathol 1986:122:36-49. Sado Y, Okigaki T, Takamiya H, et al: Experimental autoim mune glomerulonephritis with pulmonary hemorrhage in rats. The dose-effect relationship of the nephritogenic antigen from bovine glomerular basement membrane. Clin Lab Immunol 1984;15:199-204. Unanue ER, Dixon FJ: Experimental allergic glomerulonephri tis induced in the rabbit with heterologous renal antigens. J Exp Med 1967;125:149-162. Unanue ER, Dixon FJ, Feldman JD: Experimental allergic glomerulonephritis induced in the rabbit with homologous renal antigens. J Exp Med 1967:125:163-183. Couser WG, Stilmant M, Lewis EJ: Experimental glomerulone phritis in the guinea pig. I. Glomerular lesions associated with antiglomerular basement membrane antibody deposits. Lab in vest 1973:29:236-243. Couser WG, Spargo BH, Stilmant MM, et al: Experimental glomerulonephritis in the uinea pig. II. Ultrastructural lesions of the basement membrane associated with proteinuria. Lab Invest 1975:32:46-55. Steblay RW: Glomerulonephritis induced in monkeys by injec tions of heterologous glomerular basement membrane and Freund's adjuvant. Nature 1963:197:1173-1176. Bolton WK. Tucker FL, Sturgill BC: Experimental autoimmune glomerulonephritis in chickens. J Clin Lab Immunol 1980: 3:179-184. Bolton WK, Tucker FL, Sturgill BC: New avian model of experi mental glomerulonephritis consistent with mediation by cellular immunity. J Clin Invest 1984:73:1263-1276. Tucker FL, Sturgill BC, Bolton WK: Ultrastructural studies of experimental autoimmune glomerulonephritis in normal and bursectomized chickens. Lab Invest 1985;53:563-570. Bolton WK, Chandra M. Tyson TM, et al: Transfer of experi mental glomerulonephritis in chickens by mononuclear cells. Kidney lnt 1988:34:598-610. Bolton WK, Atuk NO, Sturgill BC: Nephrotoxic nephritis in rabbits. The role of the sympathetic nervous system. Am J Pathol 1978:90:689-698.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
angial cells to reach confluence. Mesangial cells stained positively for desmin while chicken fibroblasts were ne gative. By electron microscopy mesangial cells had a well-developed filamentous system and multilamellar re sidual bodies, both of which were absent in fibroblasts. Chicken mesangial cells demonstrated avid phagocytosis of polystyrene beads, bound AII and underwent contrac tion with All. Fibroblasts did not take up polystyrene beads and did not have All binding or contraction induced with All. Finally, chicken mesangial cells in culture stain with monoclonal antibody directed against chicken mesangial cell epitopes and chicken fibroblasts are negative with this monoclonal anti body [45], Mammalian mesangial cells in culture have provided us with a wealth of information relative to normal mesangial cell function. They are proving to be of use as an in vitro model to examine pathogenetic events involved in im mune and nonimmune damage to glomeruli. In the pres ent paper we have described, for the first time, the culture and characterization of chicken mesangial cells and de monstrated similarities and dissimilarities to mammalian mesangial cells. Successful culture of chicken mesangial cells should allow us to examine in vitro pathogenetic processes operative in vivo in our model of EAG in chickens. By utilization of this in vitro system, we should be able to learn more about the role of cell-mediated immunity in the pathogenesis of EAG in chickens. We should consequently learn more of the role of cell-medi ated immunity in the pathogenesis of human glomerulo nephritis.
83
84
37 Schlondorff DJ, Satriano A, Hagege J, et al: Effect of platelet activating factor and serum treated by mosan on prostaglandin E; synthesis, arachidonic acid release, and contraction of cul tured rat mesangial cells. J Clin Invest 1984:73:1227-1231. 38 Singhal PC, Ding G, DeCandido S, et al: Endocytosis by cul tured mesangial cells and associated changes in prostaglandin E: synthesis. Am J Physiol 1987:252: F627-F634. 39 Foidart J, Sraer J, Delarue F, et al: Evidence for mesangial glomerular receptors for angiotensin II linked to mesangial cell contractility. FEBS Lett 1980:121:333-339. 40 Doi TM. Striker LJ. Elliot SJ.et al: Insulinlike growth factor-1 is a progression factor for human mesangial cells. Am J Pathol 1989:134:395-404. 41 Mahieu PR, Foidart JB. DuBois CH. et al: Tissue culture of normal rat glomeruli: Contractile activity of the cultured mesan gial cells. Invest Cell Pathol 1980;3:121-128. 42 Tanaka T, Fuji wara Y, Orita A, et al : The functional characteris tics of cultured rat mesangial cell. Jpn Cire J 1984;48:1017-1029. 43 Simonson MS, Dunn MJ: Leukotriene C4 and D4 contract rat glomerular mesangial cells. Kidney Int 1986;30:524-531. 4 4 Singhal PC, Scharschmidt LA, Gibbons N, et al: Contraction and relaxation of cultured mesangial cells on a silicone rubber surface. Kidney Int 1986:30:862-873. 45 Sadovnic MJ, Bolton WK: Proof that mesangial cells in culture are truly of mesangial origin. Clin Res 1988:36:597.
Accepted: June 26,1990 W. Kline Bolton. MD Division of Nephrology Department of Internal Medicine Box 133 University of Virginia Health Sciences Center Charlottesville. VA 22908 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 9:37:09 PM
22 Dantzler WH. Braun EJ: Comparative nephron function in reptiles, birds, and mammals. Am J Physiol I980;293: R197R2I3. 23 Kreisberg Jl. Karnovsky MJ: Glomerular cells in culture. Kid ney Ini 1983;23:439-447. 24 Ausiello DA, Kreisberg J I, Roy C, et al: Contraction of cultured rat glomerular cells of apparent mesangial origin after stimula tion with angiotensin II and arginine vasopressin. J Clin Invest 1980;65:745-760. 25 Gilbert SF, Migeon BR: O-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 1975 ; 5 :11-17. 26 Striker GE, Soderland C, Bowen-Pope DF, et al: Isolation, characterization and propagation in vitro of human glomerular endothelial cells. J Exp Med 1984;160:323-328. 27 Striker GE, Striker LJ: Biology of disease: Glomerular cell culture. Lab Invest 1985:53:122-131. 28 Quadracci LJ, StrikerGE: Growth and maintenance ofglomerular cells in vitro. Proc Soc Exp Biol Med 1970:135:947-950. 29 Kreisberg. JI. Hoover RL, Karnovsky MJ: Isolation and charac terization of rat glomerular epithelial cells in vitro. Kidney Int 1978;14:21-30. 30 Scheinman JI, Fish AJ, Brown DM, et al: Human glomerular smooth muscle (mesangial) cells in culture. Lab Invest 1976; 34:150-158. 31 Norgaard JO: Cellular outgrowth from isolated glomeruli: Ori gin and characterization. Lab Invest 1983:48:526 542. 32 Kreisberg JI. Venkatachalam M, Troyer D: Contractile proper ties of cultured glomerular mesangial cells. Am J Physiol 1985:249 : F457-F463. 33 Schlondorff D: The glomerular mesangial cell: An expanding role for a specialized pericyte. FASEBJ 1987:1:272-281. 34 Sterzel RB. Lovett DH, Foellmer HG, et al: Mesangial cell Hillocks: Nodular foci of exaggerated growth of cells and matrix in prolonged culture. Am .1 Pathol 1986:125:130-140. 35 Yaoita E, Kazama T, Kawasaki K, et al: In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cell and dermal fibroblasts. Cell Pathol 1985:49:285 294. 36 Baud L, Hagege J, Sraer J, et al : Reactive oxygen production by cultured rat glomerular mesangial cells during phagocytosis is associated with stimulation of lipoxygenase activity. J Exp Med 1983:158:1836-1852.
Sadovnic/ Brand-Elnaggar/Bolton
