Journal of Dermatological Science, 2 (199 1) l-8 Elsevier
1
DESC 00054
In vivo studies of factors influencing mast cell numbers in rat skin * Beate M. Czarnetzki
and Gerd Mecklenburg
Department of Dermatology, University Clinics, Miinster, F.R. G. (Received
16 January
Key words: Mast cell; Stimulation;
1990; accepted
7 July 1990)
Growth factors; In vivo; Differentiation;
Proliferation
Abstract In order to examine the biological relevance of known in vitro stimuli for mast cell growth, the following substances were injected at two day intervals into the skin of Wistar rats: Ascaris Ag, epidermal growth factor, basic fibroblast growth factor, fibronectin, phytohemagglutinin-stimulated spleen cell supernatants, L-cell tibroblast supernatants and horse serum alone or in combination with L-cell supematants. In some experiments, rats were also injected with fresh or cultured peritoneal cells. Single injections of the different factors had no significant effect on mast cell numbers. After multiple injections (4-10 x ), deep dermal and submuscular mast cell numbers increased most markedly in ascaris sensitized animals at sites of ascaris antigen injections and in normal animals in response to a combination of horse serum and L-cell supernatants. Less pronounced increases occured with all other test substances except for epidermal growth factor which was inactive. Mast cell numbers were also increased at sites of injections of immature, cultured mast cells and less so after injections of mast cell precursors and mature cells. Taken together, these data show that in vivo growth and differentiation of cutaneous mast cells can be influenced by several fibroblastand lymphocyte-derived growth factors.
Introduction Mast cells are highly specialized resident cells of connective or mucosal tissue that play a pivotal role in anaphylactic reactions. In addition, these cells have been implicated to participate in the Correspondence to: B.M. Czarnetzki, (Present address) Department of Dermatology and Clinical Immunology, Klinikum Rudolf Virchow, Freie Universitat Berlin, Augustenburgerplatz 1, D 1000 Berlin 65, F.R.G. * Presented in part at the European Workshop of Inflammation, Basel, Switzerland (Agents Actions 19 : 344, 1986) and the meeting of the Collegium Internationale Allergologicum, Helsingborg, Sweden (Int Arch Allergy Appl Immunol 82: 259, 1987). 0923-181 l/91/$03.50
0 1991 Elsevier Science Publishers
regulation of connective tissue turnover, hematopoiesis and blood flow [ 1,2]. Increases of connective tissue mast cells have been reported to occur in and around sites of neoplastic and benign cellular proliferations and in a great variety of cutaneous inflammatory reactions, including wound healing and parasitic infestations [ 3,4]. Intestinal mucosal mast cells also increase in number during infestations with helminths [ 5,6]. Recently, further progress has been made in clarifying the potential mechanisms that control mast cell proliferation in tissues. Mucosal mast cells have been shown to be lymphocyte-, and more specifically 11-3dependent [ 71, with an augmenting effect of 11-4 [ 81. Coculture of immature, mucosal type
B.V. (Biomedical
Division)
mast cells with libroblast feeder layers has been shown to induce the maturation of these cells into connective tissue-type mast cells [ 9, lo]. In line with these findings, we have previously shown that mast cell-depleted rat peritoneal exudate cells (MCD) and subfractions of human peripheral blood monocytes can be stimulated by a culture medium supplemented with L-cell fibroblast supernatants (LCS) and horse serum (HS) to differentiate into connective tissue type mast cells in vitro [ 11,121. When LCS, HS or phytohemagglutinin (PHA)-stimulated spleen cell supernatants (PCS) were tested alone, in vitro differentiation of mast cells was less effective. Attempts to fractionate the LCS and HS in order to identify the active components have been unsuccessful until now (unpublished), and mast cell differentiation factors from Iibroblast feeder layers [ 9, lo] have so far not been identified either. The in vivo effect of potential mast cell growth factors has until now only been tested with multiple injections of IL-3 into nude mice which resulted in an intestinal mastocytosis [ 131. In the present investigation, we have attempted to confirm our own in vitro findings on mast cell differentiation [ 11,121 by injecting LCS, HS, a combination of HS/LCS and PCS into rat skin. In addition, the following agents were studied, using the same methodology: Factors that affect fibroblasts or that are derived from them, such as basic fibroblast growth factor (FGF), epidermal growth factor (EGF) and fibronectin (FN), and ascaris antigen as a model of parasite associated mast cell proliferation. In some experiments, the fate of injected mature peritoneal mast cells, of MCD, and of cultured, immature peritoneal mast cells was examined as well. The data confirm the ability of fibroblast supernatants, but also that of several of the other factors tested, to induce increases in cutaneous mast cell numbers. Injections of precultured, immature mast cells resulted in markedly increased tissue mast cells, while injections of MCD and PEC yielded less striking results.
Materials
and Methods
Reagents The following substances were obtained from their respective sources : PHA (Serva, Heidelberg, F.R.G.); EGF, basic FGF, FN (all from Collaborative Research Inc., Waltham, MA, U.S.A.); ascaris Ag (Greer Laboratories, Lenoir, NC, U.S.A.); HS and Dulbecco’s MEM (Gibco, Glasgow, U.K.). LCS were obtained from L-cell cultures that had grown to confluency, as previously described [ 141. PCS were collected from rat spleen cells following a 3-day stimulation with 0.5% PHA [15]. Cells
Fresh rat peritoneal exudate cells (PEC) were obtained after washing the peritoneal cavities of normal animals with MEM Dulbecco. The exudate contained between 8-12 y0 mast cells, the rest being lymphocytes and macrophages. MCD were obtained from rats that had received an intraperitoneal injection of 15 ml distilled water 5 days prior to cell harvest, as previously described [ 141. The MCD consisted almost exclusively of freshly immigrated macrophages, with 50.5% mast cells [ 161. All cells were washed in DMEM, counted and resuspended at 1 x 106/0.5 ml DMEM for injections. MCD were also cultured in vitro in a medium supportive of mast cell differentiation (20% LCSjl5 % HS). MCD cultured for more than 7 days consisted of z 99% mast cells at varying stages of maturation
[111Animals Male Wistar rates (E 200 g, from the Tierexperimentelles Institut, Hannover, F.R.G.) were used throughout the experiments. For injection studies, the backs of the animals were shaved, and 8 indelible ink marks were placed 1 cm lateral to the spine and 2 cm away from each other along the back. During a brief ether anesthesia, these sites were injected intradermally in duplicate with
1 ml of the following substances in MEM at 2 day intervals for up to 11 times: Ascaris antigen, 10 pi/ml, undiluted PCS, 15% HS, 20% LCS, the combination of 15% HS/20% LCS, EGF at 100 ng/ml and FN at 0.5 pg/ml. The concentrations of individual agents were chosen on the basis of previously reported efficacy in various biological systems [ 11,15,17]. For cell injection studies, 1 x lo6 cells were placed in 0.5 ml MEM. Four mm punch biopsies were taken 24 h after multiple injection, immediately after the animals had been killed. After cell injections, the test sites were biopsied 9 days later. All studies with test substances and cells were performed, using at least 6 different animals, and all experiments were repeated for at least three times. Some animals had been sensitized to ascaris antigen according to a well established method prior to the injections [ 151. Histological evaluation
Biopsies were left in the formalin fixative for no more than 24 h and were then processed routinely, using hematoxylin and eosin stains for identification of inflammatory cells. For mast cell counts, sections were left in contact with toluidine blue, pH 2.0, for 5 days in order to also allow staining of cells containing lowly sulfated glycosaminoglycans (mucosal mast cells) [ 181. Mast cell counts were performed in Toluidine blue stained sections, at 400 x magnification, passing from one field to the adjacent one, first along the upper dermis at the lower edge of the epidermis, then along the fatty layer above the muscularis, and finally along the lower edge of the muscularis in the submuscular connective tissue. The mean number of fields examined per biopsy was 14.2 k 4.1 in the upper dermis, 14.2 f 3.0 in the fatty layer and 11.4 f 4.1 in the submuscularis. Counts were performed by two independent observers. Statistical evaluation was done with the Student’s t-test, with P I 0.05 being regarded as significant.
Results In order to verify the usefulness of the present methodology, the effect of injections with ascaris antigen was investigated first. After up to 5 injections, no effect was seen in non-sensitized animals while some increases of mast cells were seen at this time in the sensitized animals (not shown). After 11 injections, increased numbers of mast cells were seen in the normal (not shown) as well as in the sensitized animals (Fig. 1). In the latter animals, increases were noted at all three locations examined. with statistical significance being
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Fig. 1. Bargraphs showing mean + 1 SD counts ofmast cells (ordinate) per microscopic field (magn. 400 x ) in the upper dermal (D), the fatty tissue (F) and the submuscular (S) locations. The animals had been injected 11 times with either ascaris antigen 0 or with medium alone m and were biopsied on day 22. * = statistically significant (P I O.OOS), compared to medium alone.
reached for dermal and fatty tissue mast cell counts. In the submuscularis, a striking mononuclear cell infiltrate was noted, with many plasma cells and up to 30% neutrophils and 23 % eosinophils (not shown). The morphology of the upper dermal mast cells was quite distinct from the mast cells in the fatty tissue and the submuscularis, and this held for all the tissues examined in this study, irrespective of the treatment given. Subepidermal mast cells were small and faintly staining (Fig. 2) whereas mast cells in the lower dermal tissues were large and stuffed with intensely staining granules (Fig. 3) which were often noted to be scattered in the extracellular connective tissue matrix.
Fig. 3. Histological section of lower rat skin, showing mast cells (arrows) in the fatty (F), the muscular (M) and the submuscular (S) tissue. The animals had been injected with HS/LCS 10 times and had been biopsied on day 20. (Orig. magn. 32 x ).
Fig. 2. Histological section of upper skin demonstrating the small, faintly staining, dendritic mast cells (arrows) in the upper dermis (D) right below the epidermis (E). The animals had been injected with HS/LCS x 6 and were biopsied on day 12. (Orig. magn. 120 x ).
After having obtained these positive data with ascaris Ag injections, agents that had previously been shown to stimulate in vitro mast cell differentiation in our laboratory were examined next. Table I summarizes the data on mast cell counts in the fatty tissue and the submuscularis after injections with PCS, 15% HS, 20% LCS and the HS/LCS mixture. More than one injection was always necessary to reach significant increases of mast cell numbers. Increased counts in response to PCS were noted in the submuscularis only, whereas HS and LCS alone yielded increases at both sites. Mast cell counts in the upper dermal region did not change with the latter 3 test agents, and the effect of LCS alone was lost after 10 injections (Table I).
TABLE
I
Percentage increase of mast cells in fatty layer and submuscularis HS/LCS, compared to medium-injected sites Number of injections
1 4 10 * = statistically
Fat
Submusc.
Fat
Submusc.
Fat
Submusc.
Fat
Submusc.
0 44.2 21.8
38.4 118.6* 225.1%
0 6.9 36.6*
0 240.0’ 124.0*
23.4 104.4* 0.0
0 212.6* 35.7
26.8 143.5* 103.1*
31.9 130.0* 175.0*
significant, P < 0.05.
jections, with statistical significance only in the submuscular region. Infiltrates of mononuclear cells with up to 19 y0 eosinophils and 43 y0 neutrophils were seen in the submuscularis already after 5 injections with all three test agents. In a final set of experiments, the effect of cell injections was examined. The data are sum-
20. 1816-
II
Mean f standard deviation of mast cell counts in fatty tissue and submuscularis after 10 injections of EGF, FGF, FN or medium (C)
EGF FGF FN C
of PCS HS, LCS and
HS/LCS
LCS
HS
PCS
With the 15 % HS/20% LCS mixture, results were not consistent. Statistically significant differences were observed for both the increases of mast cells in fatty tissue and the submuscularis from 4 injections onward (Table I). A significant increase in upper dermal mast cells was observed in only one specimen after 6 injections (Fig. 2). After 2 1 injection of either of the 4 test substances, a mild inflammatory infiltrate was seen in the deep cutaneous tissue which developed into a striking submuscular inflammatory infiltrate after 10 injections (not shown) and which resembled the one induced by the ascaris antigen (see above). In the subsequent experiments, the effect of two purified growth factors and of FN were examined. Results are shown in Table II. EGF was ineffective whereas FGF and FN caused increases of mast cell numbers only after 10 in-
TABLE
after different numbers of injections
Fatty tissue
Submuscularis
5.11 11.67 14.38 9.08
6.71 17.30 14.29 8.17
i_ 2.08 k 3.09 f 8.26 + 4.21
& 3.15 * 4.37* k 5.80* + 2.48
Lower numbers of injection caused no statistically significant increases (*P < 0.025).
6 1
S
Fig. 4. Mean f 1 SD of mast cell counts (ordinate) in the upper dermal (D), the fatty tissue (F) and the submuscular (S) regions after injections of either PEC 0, MCD I, 1 week old cultured MCD m or medium alone m. Biopsies were taken 9 days after a single cell injection. * = significantly different compared to medium control (PI 0.05).
b
marized in Fig. 4. In the submuscularis, all three cell preparations induced statistically significant increases of mast cells, compared to medium alone. At injection sites of cultured MCD, there was also an increase in upper dermal and fatty tissue mast cells. Injections of more mature mast cells derived from MCD after 2 2 weeks of culture were however ineffective (not shown). A mononuclear infiltrate was seen in the submuscularis after injections of all cell preparations. Discussion The present data confirm our previous in vitro data [ 1 l] which showed that a combination of HS/LCS constituted the most effective stimulus for mast cell differentiation. The findings after injections of the individual components of this mixture and of PCS were less striking. The data also confirm that parasitic antigens belong to the most potent stimuli for mast cell proliferation since injections of ascaris antigen caused not only increases of the deep dermal and the submuscular, but also of the small subepidermal mast cells (Fig. 2). It has recently been shown that parasitic antigens trigger an IgE receptor-dependent in vitro proliferation of sensitized mouse connective tissue mast cells [ 191. This might explain the in vivo effects observed here with ascaris antigen, although growth and differentiation factors generated from other cells might also be involved directly or indirectly by stimulating the immigration and maturation of mast cell precursors. The current studies also yield a number of new insights. They show that multiple injections of FGF and FN induced a moderate degree of deep dermal mast cell proliferation whereas EGF was ineffective at the concentration tested (Table II). The lack of a response of the small upper dermal mast cells to these agents may be due to a differential sensitivity of this mast cell subtype to different growth factors or to a lack of diffusion of the factors from the site of injection in the deeper cutaneous compartments up into the subepiderma1 region. The time course for increases in mast cell numbers was roughly parallel for all test sub-
stances, including the ascaris antigen, and it resembled that observed during in vitro cultures
[ill. While it is likely that the PCS contains 11-3,the factors inducing in vitro and in vivo mast cell differentiation in HS and LCS are unknown and need to be identified. Since this has proven to be difficult in the past, pure, commercially available growth factors were examined instead. EGF is known to induce fibroblast proliferation and migration [ 20,211 and FGF stimulates endothelial and 3T3 fibroblast growth and differentiation [22]. FN is produced by many cell types, including fibroblasts, and it also induces fibroblast proliferation, chemotaxis and differentiation of these cells [ 23-251. On the basis of their ability to affect fibroblast function and numbers, it was thus conceivable that these factors might also influence mast cell differentiation and proliferation in the tissue. They might also or alternatively recruit mast cell precursors from the circulation. The present findings show that both FGF and FN but not EGF influence mast cell numbers in vivo. Whether this is due to a direct effect on the mast cells and their precursors or to an indirect action via the fibroblasts, needs to be further clarified, also by in vitro studies. Since all three factors induced an inflammatory infiltrate, its presence alone seems not to be a prerequsite for increased mast cell numbers in the tissues. In previous in vitro investigations, we have shown that MCD and to a lesser extent PEC contain mast cell precursors which can be induced to differentiate in vitro after appropriate stimulation [ 111. This has been confirmed by others who showed that PEC contain five times more mast cell precursors than bone marrow cells and that one out of 17 of these cells can make mast cell clusters after injection into the skin of mice [26]. The present findings after cell injections (Fig. 4) suggest that young mast cells, at a certain stage of their in vitro development, can be further nurtured in vivo to develop into mature cells in a much- more efficient way than their immature precursors in MCD and PEC preparafactors that initiate and tions. Apparently,
7
maintain mast cell differentiation are not present at sufficiently high level in normal tissue to induce a significant stimulation of the injected precursors in the MCD and PEC exudates. The lack of a marked increase of mast cell numbers at sites of PEC and mature cultured mast cell injections could possibly be due to an inability of mature mast cells to survive in vivo due to the trauma during in vitro handling. An additional possibility would be an inhibition of mast cell precursor recruitment from the blood or their local proliferation and differentiation by mature mast cells [27]. We have observed this during our in vitro studies with both fresh mast cells in the PEC and with old, relatively mature cultured cells [ 111. The same possibility has previously been suggested by other authors [28]. In summary, these in vivo findings support the concept that mast cell differentiation and proliferation is a complex process that depends on a number of factors for support. These factors will have to be further identified, and their specific contribution to mast cell growth and development will have to be elucidated in more detail.
10
11
Acknowledgements 12
Supported by grant Cz 22/l-5 of the German Research Foundation (DFG). 13
References Galli SJ, Dvorak AM, Dvorak HF: Basophils and mast cells: morphologic insights into their biology. Prog Allergy 34: 1-141, 1984. Marks RM, Roche WR, Czerniecki M, Penny R, Nelson DS: Mast cell granules cause proliferation of human microvascular endothelial cells. Lab Invest 55: 298-294, 1986. Asboe-Hansen G: A survey of the normal and pathological occurrence of mutinous substances and mast cells in the dermal connective tissue in man. Acta derm-vener Stockh 30: 338-347, 1950. Matsuda H, Kitamura Y: Migration of stromal cells supporting mast-cell differentiation into open wound produced in the skin of mice. Exp Hematol 9: 38-43, 1981. Mayrhofer G: The nature of the thymus dependency of
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15
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mucosal mast cells. I. An adaptive secondary response to challenge with Nippostrongylus brasiliensis. Cell Immunol47: 304-311, 1979. Parmentier HK, Ruitenberg EJ, Elgersma A: Thymus dependency of the adoptive transfer of intestinal mastocytopoiesis in Trichinella spiralis-infected mice. Int Archs Allergy appl Immunol 68: 260-267, 1982. Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Firch F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynski E, Dy M, Lebel B: Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHIgrowth factor activity, mast cell growth factor activity, P cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J Immunol 131: 282-287, 1983. Schmitt E, Fassbender B, Beyreuther K, Spaeth E, Schwarzkopf R, Rude E: Characterization of a T cellderived lymphokine that acts synergistically with 113 on the growth of murine mast cells and is identical with I1 4. lmmunobiol 174: 406-419, 1987. Davidson S, Mansour A, Gallily R, Smolarski M, Rodolovitch M, Ginsburg H: Mast cell differentiation depends on T cells and granule synthesis on libroblasts. Immunology 48: 439-452, 1983. Levi-Schaffer F, Austen KF, Gravallese PM, Stevens RL: Coculture of interleukin 3-dependent mouse mast cells with libroblasts results in a phenotypic change of the mast cells. Proc Nat1 Acad Sci USA 83: 6485-6488,1986. Czarnetzki BM, Sterry W, Bazin H, Kalveram K: Evidence that tissue mast cells derive from mononuclear phagocytes. Int Archs Allergy Appl Immuno167: 44-48, 1983. Czarnetzki BM, Figdor CG, Kolde G, Vroom T, Aalberse R, De Vries J: Development of connective tissue mast cells from purified blood monocytes. Immunology 51: 549-554, 1984. Abe T, Ochiai H, Minamishima Y, Nawa Y: Induction of intestinal mastocytosis in nude mice by repeated injection of interleukin-3. Int Archs Allergy Appl Immunol 86: 356-358, 1988. Czarnetzki BM, Hannich D, Niedorf H: In-vitro studies of rat peritoneal mast cells. Immunobiol 156: 470-476, 1979. Czarnetzki BM, Wiillenweber I: In vitro migratory response of rat peritoneal macrophages and mast cells towards chemotactic factors and growth factors. J Invest Dermatol 91: 224-227, 1988. Sterry W, Czarnetzki BM: In vitro differentiation of rat peritoneal macrophages into mast cells: An enzymecytochemical study. Blut 44: 211-220, 1982. Johnson HM, Torres BA: Peptide Growth factors PDGF, EGF, and FGF regulate interferon-y production. J Immunol 134: 2824-2826, 1985. Wingren U, Enerblck L: Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining prop-
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20 21
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erties, with special reference to protein blocking and solubility of the granular glycosaminoglycans. Histochem J 15: 571-582, 1983. Takagi M, Nakahata K, Koike K, Kobayashi T, Tsuji K, Kojima S, Hirano T, Miyajima A, Arai K, Akabane T: Stimulation of connective tissue-type mast cell proliferation by crosslinking of cell-bound IgE. J Exp Med 170: 233-244, 1989. Carpenter G: Epidermal growth factor. Ann Rev Biochem 48: 193-216, 1979. Westermark B, Blomquist E: Stimulation of tibroblast migration by epidermal growth factor. Cell Biol Int Rep 4: 649-652, 1980. Gospodarowicz D: Isolation and characterization of acidic and basic tibroblast growth factor, in: Methods in enzymology. Peptide growth factors. Edited by D Barnes Orlando and DA Sirbaska, 1987, Academic Press Inc., pp 106-119. Vartio T: Fibronectin: Multiple interactions assigned to structural domains. Med Biol 61: 283-295, 1981.
24 Tsukamoto Y, Helsel, WE, Wahl SM: Macrophage production of fibronectin, a chemoattractant for tibroblasts. J Immunol 127: 673-678, 1981. 25 Mosher DF, Furcht LT: Fibronectin: Review of its structure and possible functions. J Invest Dermatol 77: 175-180, 1981. 26 SonadaT, KanayamaY, Hara H., Hayashi C, Tadokoro M, Yonezawa T, Kitamura Y: Proliferation of peritoneal mast cells in the skin of W/W” mice that genetically lack mast cells. J Exp Med 160: 138-151, 1984. 27 Kanakura Y, Kuriu A, Waki N, Nakano T, Asai H, YonezawaT, KitamuraY: Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrowderived percursors. Blood 71: 573-580, 1988. 28 Kitamura Y, Nakano T, Kanakura Y, Matsuda H: Factors influencing mast cell differentiation. Proc XII Int Congr Allergol Clin Immunol. Philadelphia, 1986, CV Mosby pp 154-157, 1986.
1
DESC 00054
In vivo studies of factors influencing mast cell numbers in rat skin * Beate M. Czarnetzki
and Gerd Mecklenburg
Department of Dermatology, University Clinics, Miinster, F.R. G. (Received
16 January
Key words: Mast cell; Stimulation;
1990; accepted
7 July 1990)
Growth factors; In vivo; Differentiation;
Proliferation
Abstract In order to examine the biological relevance of known in vitro stimuli for mast cell growth, the following substances were injected at two day intervals into the skin of Wistar rats: Ascaris Ag, epidermal growth factor, basic fibroblast growth factor, fibronectin, phytohemagglutinin-stimulated spleen cell supernatants, L-cell tibroblast supernatants and horse serum alone or in combination with L-cell supematants. In some experiments, rats were also injected with fresh or cultured peritoneal cells. Single injections of the different factors had no significant effect on mast cell numbers. After multiple injections (4-10 x ), deep dermal and submuscular mast cell numbers increased most markedly in ascaris sensitized animals at sites of ascaris antigen injections and in normal animals in response to a combination of horse serum and L-cell supernatants. Less pronounced increases occured with all other test substances except for epidermal growth factor which was inactive. Mast cell numbers were also increased at sites of injections of immature, cultured mast cells and less so after injections of mast cell precursors and mature cells. Taken together, these data show that in vivo growth and differentiation of cutaneous mast cells can be influenced by several fibroblastand lymphocyte-derived growth factors.
Introduction Mast cells are highly specialized resident cells of connective or mucosal tissue that play a pivotal role in anaphylactic reactions. In addition, these cells have been implicated to participate in the Correspondence to: B.M. Czarnetzki, (Present address) Department of Dermatology and Clinical Immunology, Klinikum Rudolf Virchow, Freie Universitat Berlin, Augustenburgerplatz 1, D 1000 Berlin 65, F.R.G. * Presented in part at the European Workshop of Inflammation, Basel, Switzerland (Agents Actions 19 : 344, 1986) and the meeting of the Collegium Internationale Allergologicum, Helsingborg, Sweden (Int Arch Allergy Appl Immunol 82: 259, 1987). 0923-181 l/91/$03.50
0 1991 Elsevier Science Publishers
regulation of connective tissue turnover, hematopoiesis and blood flow [ 1,2]. Increases of connective tissue mast cells have been reported to occur in and around sites of neoplastic and benign cellular proliferations and in a great variety of cutaneous inflammatory reactions, including wound healing and parasitic infestations [ 3,4]. Intestinal mucosal mast cells also increase in number during infestations with helminths [ 5,6]. Recently, further progress has been made in clarifying the potential mechanisms that control mast cell proliferation in tissues. Mucosal mast cells have been shown to be lymphocyte-, and more specifically 11-3dependent [ 71, with an augmenting effect of 11-4 [ 81. Coculture of immature, mucosal type
B.V. (Biomedical
Division)
mast cells with libroblast feeder layers has been shown to induce the maturation of these cells into connective tissue-type mast cells [ 9, lo]. In line with these findings, we have previously shown that mast cell-depleted rat peritoneal exudate cells (MCD) and subfractions of human peripheral blood monocytes can be stimulated by a culture medium supplemented with L-cell fibroblast supernatants (LCS) and horse serum (HS) to differentiate into connective tissue type mast cells in vitro [ 11,121. When LCS, HS or phytohemagglutinin (PHA)-stimulated spleen cell supernatants (PCS) were tested alone, in vitro differentiation of mast cells was less effective. Attempts to fractionate the LCS and HS in order to identify the active components have been unsuccessful until now (unpublished), and mast cell differentiation factors from Iibroblast feeder layers [ 9, lo] have so far not been identified either. The in vivo effect of potential mast cell growth factors has until now only been tested with multiple injections of IL-3 into nude mice which resulted in an intestinal mastocytosis [ 131. In the present investigation, we have attempted to confirm our own in vitro findings on mast cell differentiation [ 11,121 by injecting LCS, HS, a combination of HS/LCS and PCS into rat skin. In addition, the following agents were studied, using the same methodology: Factors that affect fibroblasts or that are derived from them, such as basic fibroblast growth factor (FGF), epidermal growth factor (EGF) and fibronectin (FN), and ascaris antigen as a model of parasite associated mast cell proliferation. In some experiments, the fate of injected mature peritoneal mast cells, of MCD, and of cultured, immature peritoneal mast cells was examined as well. The data confirm the ability of fibroblast supernatants, but also that of several of the other factors tested, to induce increases in cutaneous mast cell numbers. Injections of precultured, immature mast cells resulted in markedly increased tissue mast cells, while injections of MCD and PEC yielded less striking results.
Materials
and Methods
Reagents The following substances were obtained from their respective sources : PHA (Serva, Heidelberg, F.R.G.); EGF, basic FGF, FN (all from Collaborative Research Inc., Waltham, MA, U.S.A.); ascaris Ag (Greer Laboratories, Lenoir, NC, U.S.A.); HS and Dulbecco’s MEM (Gibco, Glasgow, U.K.). LCS were obtained from L-cell cultures that had grown to confluency, as previously described [ 141. PCS were collected from rat spleen cells following a 3-day stimulation with 0.5% PHA [15]. Cells
Fresh rat peritoneal exudate cells (PEC) were obtained after washing the peritoneal cavities of normal animals with MEM Dulbecco. The exudate contained between 8-12 y0 mast cells, the rest being lymphocytes and macrophages. MCD were obtained from rats that had received an intraperitoneal injection of 15 ml distilled water 5 days prior to cell harvest, as previously described [ 141. The MCD consisted almost exclusively of freshly immigrated macrophages, with 50.5% mast cells [ 161. All cells were washed in DMEM, counted and resuspended at 1 x 106/0.5 ml DMEM for injections. MCD were also cultured in vitro in a medium supportive of mast cell differentiation (20% LCSjl5 % HS). MCD cultured for more than 7 days consisted of z 99% mast cells at varying stages of maturation
[111Animals Male Wistar rates (E 200 g, from the Tierexperimentelles Institut, Hannover, F.R.G.) were used throughout the experiments. For injection studies, the backs of the animals were shaved, and 8 indelible ink marks were placed 1 cm lateral to the spine and 2 cm away from each other along the back. During a brief ether anesthesia, these sites were injected intradermally in duplicate with
1 ml of the following substances in MEM at 2 day intervals for up to 11 times: Ascaris antigen, 10 pi/ml, undiluted PCS, 15% HS, 20% LCS, the combination of 15% HS/20% LCS, EGF at 100 ng/ml and FN at 0.5 pg/ml. The concentrations of individual agents were chosen on the basis of previously reported efficacy in various biological systems [ 11,15,17]. For cell injection studies, 1 x lo6 cells were placed in 0.5 ml MEM. Four mm punch biopsies were taken 24 h after multiple injection, immediately after the animals had been killed. After cell injections, the test sites were biopsied 9 days later. All studies with test substances and cells were performed, using at least 6 different animals, and all experiments were repeated for at least three times. Some animals had been sensitized to ascaris antigen according to a well established method prior to the injections [ 151. Histological evaluation
Biopsies were left in the formalin fixative for no more than 24 h and were then processed routinely, using hematoxylin and eosin stains for identification of inflammatory cells. For mast cell counts, sections were left in contact with toluidine blue, pH 2.0, for 5 days in order to also allow staining of cells containing lowly sulfated glycosaminoglycans (mucosal mast cells) [ 181. Mast cell counts were performed in Toluidine blue stained sections, at 400 x magnification, passing from one field to the adjacent one, first along the upper dermis at the lower edge of the epidermis, then along the fatty layer above the muscularis, and finally along the lower edge of the muscularis in the submuscular connective tissue. The mean number of fields examined per biopsy was 14.2 k 4.1 in the upper dermis, 14.2 f 3.0 in the fatty layer and 11.4 f 4.1 in the submuscularis. Counts were performed by two independent observers. Statistical evaluation was done with the Student’s t-test, with P I 0.05 being regarded as significant.
Results In order to verify the usefulness of the present methodology, the effect of injections with ascaris antigen was investigated first. After up to 5 injections, no effect was seen in non-sensitized animals while some increases of mast cells were seen at this time in the sensitized animals (not shown). After 11 injections, increased numbers of mast cells were seen in the normal (not shown) as well as in the sensitized animals (Fig. 1). In the latter animals, increases were noted at all three locations examined. with statistical significance being
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8-
F
Fig. 1. Bargraphs showing mean + 1 SD counts ofmast cells (ordinate) per microscopic field (magn. 400 x ) in the upper dermal (D), the fatty tissue (F) and the submuscular (S) locations. The animals had been injected 11 times with either ascaris antigen 0 or with medium alone m and were biopsied on day 22. * = statistically significant (P I O.OOS), compared to medium alone.
reached for dermal and fatty tissue mast cell counts. In the submuscularis, a striking mononuclear cell infiltrate was noted, with many plasma cells and up to 30% neutrophils and 23 % eosinophils (not shown). The morphology of the upper dermal mast cells was quite distinct from the mast cells in the fatty tissue and the submuscularis, and this held for all the tissues examined in this study, irrespective of the treatment given. Subepidermal mast cells were small and faintly staining (Fig. 2) whereas mast cells in the lower dermal tissues were large and stuffed with intensely staining granules (Fig. 3) which were often noted to be scattered in the extracellular connective tissue matrix.
Fig. 3. Histological section of lower rat skin, showing mast cells (arrows) in the fatty (F), the muscular (M) and the submuscular (S) tissue. The animals had been injected with HS/LCS 10 times and had been biopsied on day 20. (Orig. magn. 32 x ).
Fig. 2. Histological section of upper skin demonstrating the small, faintly staining, dendritic mast cells (arrows) in the upper dermis (D) right below the epidermis (E). The animals had been injected with HS/LCS x 6 and were biopsied on day 12. (Orig. magn. 120 x ).
After having obtained these positive data with ascaris Ag injections, agents that had previously been shown to stimulate in vitro mast cell differentiation in our laboratory were examined next. Table I summarizes the data on mast cell counts in the fatty tissue and the submuscularis after injections with PCS, 15% HS, 20% LCS and the HS/LCS mixture. More than one injection was always necessary to reach significant increases of mast cell numbers. Increased counts in response to PCS were noted in the submuscularis only, whereas HS and LCS alone yielded increases at both sites. Mast cell counts in the upper dermal region did not change with the latter 3 test agents, and the effect of LCS alone was lost after 10 injections (Table I).
TABLE
I
Percentage increase of mast cells in fatty layer and submuscularis HS/LCS, compared to medium-injected sites Number of injections
1 4 10 * = statistically
Fat
Submusc.
Fat
Submusc.
Fat
Submusc.
Fat
Submusc.
0 44.2 21.8
38.4 118.6* 225.1%
0 6.9 36.6*
0 240.0’ 124.0*
23.4 104.4* 0.0
0 212.6* 35.7
26.8 143.5* 103.1*
31.9 130.0* 175.0*
significant, P < 0.05.
jections, with statistical significance only in the submuscular region. Infiltrates of mononuclear cells with up to 19 y0 eosinophils and 43 y0 neutrophils were seen in the submuscularis already after 5 injections with all three test agents. In a final set of experiments, the effect of cell injections was examined. The data are sum-
20. 1816-
II
Mean f standard deviation of mast cell counts in fatty tissue and submuscularis after 10 injections of EGF, FGF, FN or medium (C)
EGF FGF FN C
of PCS HS, LCS and
HS/LCS
LCS
HS
PCS
With the 15 % HS/20% LCS mixture, results were not consistent. Statistically significant differences were observed for both the increases of mast cells in fatty tissue and the submuscularis from 4 injections onward (Table I). A significant increase in upper dermal mast cells was observed in only one specimen after 6 injections (Fig. 2). After 2 1 injection of either of the 4 test substances, a mild inflammatory infiltrate was seen in the deep cutaneous tissue which developed into a striking submuscular inflammatory infiltrate after 10 injections (not shown) and which resembled the one induced by the ascaris antigen (see above). In the subsequent experiments, the effect of two purified growth factors and of FN were examined. Results are shown in Table II. EGF was ineffective whereas FGF and FN caused increases of mast cell numbers only after 10 in-
TABLE
after different numbers of injections
Fatty tissue
Submuscularis
5.11 11.67 14.38 9.08
6.71 17.30 14.29 8.17
i_ 2.08 k 3.09 f 8.26 + 4.21
& 3.15 * 4.37* k 5.80* + 2.48
Lower numbers of injection caused no statistically significant increases (*P < 0.025).
6 1
S
Fig. 4. Mean f 1 SD of mast cell counts (ordinate) in the upper dermal (D), the fatty tissue (F) and the submuscular (S) regions after injections of either PEC 0, MCD I, 1 week old cultured MCD m or medium alone m. Biopsies were taken 9 days after a single cell injection. * = significantly different compared to medium control (PI 0.05).
b
marized in Fig. 4. In the submuscularis, all three cell preparations induced statistically significant increases of mast cells, compared to medium alone. At injection sites of cultured MCD, there was also an increase in upper dermal and fatty tissue mast cells. Injections of more mature mast cells derived from MCD after 2 2 weeks of culture were however ineffective (not shown). A mononuclear infiltrate was seen in the submuscularis after injections of all cell preparations. Discussion The present data confirm our previous in vitro data [ 1 l] which showed that a combination of HS/LCS constituted the most effective stimulus for mast cell differentiation. The findings after injections of the individual components of this mixture and of PCS were less striking. The data also confirm that parasitic antigens belong to the most potent stimuli for mast cell proliferation since injections of ascaris antigen caused not only increases of the deep dermal and the submuscular, but also of the small subepidermal mast cells (Fig. 2). It has recently been shown that parasitic antigens trigger an IgE receptor-dependent in vitro proliferation of sensitized mouse connective tissue mast cells [ 191. This might explain the in vivo effects observed here with ascaris antigen, although growth and differentiation factors generated from other cells might also be involved directly or indirectly by stimulating the immigration and maturation of mast cell precursors. The current studies also yield a number of new insights. They show that multiple injections of FGF and FN induced a moderate degree of deep dermal mast cell proliferation whereas EGF was ineffective at the concentration tested (Table II). The lack of a response of the small upper dermal mast cells to these agents may be due to a differential sensitivity of this mast cell subtype to different growth factors or to a lack of diffusion of the factors from the site of injection in the deeper cutaneous compartments up into the subepiderma1 region. The time course for increases in mast cell numbers was roughly parallel for all test sub-
stances, including the ascaris antigen, and it resembled that observed during in vitro cultures
[ill. While it is likely that the PCS contains 11-3,the factors inducing in vitro and in vivo mast cell differentiation in HS and LCS are unknown and need to be identified. Since this has proven to be difficult in the past, pure, commercially available growth factors were examined instead. EGF is known to induce fibroblast proliferation and migration [ 20,211 and FGF stimulates endothelial and 3T3 fibroblast growth and differentiation [22]. FN is produced by many cell types, including fibroblasts, and it also induces fibroblast proliferation, chemotaxis and differentiation of these cells [ 23-251. On the basis of their ability to affect fibroblast function and numbers, it was thus conceivable that these factors might also influence mast cell differentiation and proliferation in the tissue. They might also or alternatively recruit mast cell precursors from the circulation. The present findings show that both FGF and FN but not EGF influence mast cell numbers in vivo. Whether this is due to a direct effect on the mast cells and their precursors or to an indirect action via the fibroblasts, needs to be further clarified, also by in vitro studies. Since all three factors induced an inflammatory infiltrate, its presence alone seems not to be a prerequsite for increased mast cell numbers in the tissues. In previous in vitro investigations, we have shown that MCD and to a lesser extent PEC contain mast cell precursors which can be induced to differentiate in vitro after appropriate stimulation [ 111. This has been confirmed by others who showed that PEC contain five times more mast cell precursors than bone marrow cells and that one out of 17 of these cells can make mast cell clusters after injection into the skin of mice [26]. The present findings after cell injections (Fig. 4) suggest that young mast cells, at a certain stage of their in vitro development, can be further nurtured in vivo to develop into mature cells in a much- more efficient way than their immature precursors in MCD and PEC preparafactors that initiate and tions. Apparently,
7
maintain mast cell differentiation are not present at sufficiently high level in normal tissue to induce a significant stimulation of the injected precursors in the MCD and PEC exudates. The lack of a marked increase of mast cell numbers at sites of PEC and mature cultured mast cell injections could possibly be due to an inability of mature mast cells to survive in vivo due to the trauma during in vitro handling. An additional possibility would be an inhibition of mast cell precursor recruitment from the blood or their local proliferation and differentiation by mature mast cells [27]. We have observed this during our in vitro studies with both fresh mast cells in the PEC and with old, relatively mature cultured cells [ 111. The same possibility has previously been suggested by other authors [28]. In summary, these in vivo findings support the concept that mast cell differentiation and proliferation is a complex process that depends on a number of factors for support. These factors will have to be further identified, and their specific contribution to mast cell growth and development will have to be elucidated in more detail.
10
11
Acknowledgements 12
Supported by grant Cz 22/l-5 of the German Research Foundation (DFG). 13
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