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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 14(3), 625-635 (1992)
Chemotactic Activities of Peripheral Blood Polymorphonuclear Leukocytes and Peritoneal Exudate Polymorphonuclear Leukocytes in MRL Mice. Sumi ko Sasagawa', Yukio Satow*, Kazuo Suzuki3, Tomokazu Hosokawa4 'Radiation Effects Research Foundation, Hiroshima 732, Japan 2Hiroshima University, Hiroshima 734, Japan 3Ja~anNational Institue of Health, Tokyo 141, Japan 4Kyoto Prefectural University of Medicine, Kyoto 606, Japan
ABSTRACT Chemotactic responsiveness to fMet-Leu-Phe in concentrations of to 10-4M in the Boyden chamber was compared between peritoneal exudate cells (PEC) and polymorphonuclear leukocytes (PMN) isolated from peripheral blood, between MRL/Mp-+/+ (MRL-+/+) and MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice, and between young (6 - 9 week old) and aged (16 - 24 week old) mice. Chemotactic responsiveness of PEC did not differ between MRL-+/+ and MRL-lpr/lpr, and young and aged mice. While, PMN shored greater chemotaxis in aged MRL-+/+ mice than that in aged MRL-lpr/lpr mice. These results suggest that chemotactic responsiveness of PMN differ from that of PEC which is assumed to be preactivated by an inflammatory agent injected into the peritoneal cavity to elicit cells. Less responsiveness of PMN to the bacterial origin peptide might relate to the autoimmune disease of this murine model.
I NTRODUCTI ON Pol ymorphonuclear leukocytes (PMN) are essential for normal host defenses by virtue of their ability to seek out, recognize, ingest and ki 1 1 microorganisms. However, their excessively infIammatory response is 625 Copyright 0 1992 by Marcel Dekker, Inc.
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thought to be harmful. Human autoimmune diseases develop a lot of various forms of' tissue injury. In rheumatoid arthritis, a chronic state of inflammation exists i n which the synovial fluid often contains a large number of PMN (1,2). Accumulation and hyperfunction of PMN in inflammatory sites including release of lysosomal enzymes and product ion of superoxide anions are contributable to the pathogenesis of the disease (3-7). While, infection is a major cause of morbidity and mortality among patients with systemic lupus erythematosus (SLE) (8,9). Defect of PMN chemotactic responsiveness have been identified i n patients with incr eased susceptibi 1 i ty to recurrent bacterial infections,including patien ts with SLE (10, 11). MRL mice are a murine model that spontaneously develops autoimmune disease (12). MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice are we1 1 known to involve an autosomal recessive mutant genome Ipr, which produces massive T-cell proliferation and early onset of autoimmune diseases and early death with arthritis and glomerulonephritis during their second year of life (13). A I 1 MRL- I pr/ 1 pr mice deve 1op mass i ve genera I i zed 1ymph node en 1argement, etc., and early death with glomerulonephritis. MRL/Mp-t/+ (MRL-+/t) mice are an unaffected substrain which do not develop lymphoprol iferative syndrome. Their immunity and pathology have been we1 1 studied. However, PMN which are thought to play a crucial role i n the pathophysiology of the autoimmune diseases have not yet been studied in this type of mice, because of a lack of suitable methods for studying murine PMN. Human PMN are frequently isolated for studies from peripheral blood, however,the small volume of blood from mice makes this a difficult source when study i ng mur i ne PMN. Therefore many studies have used per i tonea I exudate cells (PEC) elicited by the injection of an inflammatory agent. Our preliminary trials showed that PEC of mice showed markedly enhanced superoxide anion production, suggesting that PEC had already been activated by an inflammatory procedure. In the present study, we examined migrating activity of PEC and PMN obtained from MRL-lpr/lpr and MRL-t/t mice.
CHEMOTACTIC RESPONSE OF PMN
621
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MATERIALS AND METHODS Mice : MRL-+/+ and MRL-lpr/lpr mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained at our animal facility. Six t o eight week-old mice were used as the young group and 16 to 24 week old mice as the aged group. Preparation of PEC : This procedure was done according t o the method of Yamashita et a1 (14,151 with a slight modification. PEC highly enriched for inflammatory PMN were induced by intraperitoneal injection of one ml of 15% glycogen. Six hours later, mice were killed by cervical dislocation and the peritoneal cavities were washed out. PEC were collected by lavage of the peritoneum with a 5 ml of PBS containing 5 U/ml of heparin. After removal of containing erythrocytes by a hypotonic solution of a 0.75% ammonium chloride (supplemented by 20mM Tris-HCI, PH cells were twice washed with phosphate buffered saline (PBS) by centrifugation at 400 g for 10 min at 2 O O C . 7.4),
Preparation of PMN : Heparinized (20 W m l ) peripheral blood samples obtained from three to four mice were pooled and mixed with 1.5% (W/V> dextran T-500 - PBS solution. Containing erythrocytes in the leukocyte fraction were removed by lysis with a 0.75% ammonium chloride solution containing 20 mM Tris-HCI (final pH 7.4) for 5 min at 37°C with gentle shaking. PMN enriched fraction was obtained by a centrifugation of the discontinuous density gradient of Percoll. Percoll Pharmac i a Fine Chemicals, Uppsala, Sweden) was diluted as described by Harbeck et al (16). A stock solution labelled 100% was prepared by m Xing 9 volumes of Percoll with 1 volume of 10 x PBS. The stock solut on was diluted to solutions of 65%, 60%, and 55% Percoll. Preformed gradients of 6 ml were Prepared in disposable polystyrene tubes (120 by 16 mm; Corning). Pasteur Pipettes were used to layer successively 2 ml of each of the 55, and 60% Percoll solutions onto 2 ml of the 65% solution. Finally, one ml of the leukocyte suspension (approximately 5 x lo6 cells) were loaded onto each
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628
SASAGAWA ET A L .
gradient. Tubes were centrifuged at 1600 g for 30 min at 4OC before harvest of each cell band with Pasteur pipettes. Harvested cells on the 60% Percoll laver were twice washed with PBS. Cel I counts : Differential leukocyte counts were made by examination of Wright-stained preparations of the pel leted cel Is which were smeared on a slide glass. After staining, the smeared slide glass was read under transmitted brigh field (x 40) and the proportion of granulocytic leukocytes to tot 1 leukocytes were recorded. In calculating this percentage,broken cel Is were not included. A minimum of 200 cells were counted for each smear. Percentages of granulocytic leukocytes in Wright stain were around 75 - 90% for PEC, 15 - 20% for peripheral blood leukocytes, and 65-75% for the PMN enriched fraction. These percentage values were always shown through the experiments. Measurement of migrating activity : Migration was measured using the modified Boyden chamber method (17). Cells were suspended at a concentration of 2 x 106 cells/ml in Hanks balanced salt solution (HBSS) that contained 0.9% bovine serum albumin and 0.27% NaHC03, and were incubated in a Boyden chamber (Bio-Rad Lab, Richmond, CAI for 90 min at 37°C under 5% Con. The migration distance (pm/90 m i d from the surface of a Mi 1 1 ipore f i lter (3.0 pm pore size, Mi 1 1 ipore, Bedford, MA, U. S . A. 1 The resulting t o the leading front of the cells i n it was measured (18). value was used to determine random migration and chemotaxis to fMet-Leu-Phe (FMLP).
RESULTS Chemotactic activity of PEG FIG 1 demonstrates chemotactic activity of PEC in MRL mice. PEC obtained both from MRL-lpr/lpr and MRL-+/+ mice migrated toward FMLP ranging from to 10-4M in a concentration dependent manner. The
629
CHEMOTACTIC RESPONSE OF PMN
2 80
r
HRL-
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0 0)
w z U
2 4 0
720
I?
E
O
L
0
I 1
10-8
I
I
I
10-7 10-6 10-5 CONCENTRATION ( M 1
1
10-4
0
L ’ 0
‘10-8
I
I
I
10-7 10-6 10-5 CONCENTRATION ( M 1
I
10-4
FIG 1. Chemotactic activities of PEC in MRL mice. PEC in a Boyden chamber were incubated for 90 min at 37OC under 5% C02. The distance that PEC migrated from the surface into a 3.0 pm pore diameter Millipore filter was measured. FMLP in concentrations of 10-8M to 10-4M were added to the lower compartments of the chamber. Each point and vertical bar represents the mean and SE for 6 to 8 mice. 0-- - : young males, 0 - - - : young females, 0- : aged males, 0: aged females.
chernotactic activities did not differ between male and female mice, nor between young and aged mice except at concentrations of 10-*M and 10-6M of FMLP, where chemotactic distances in aged female MRL-lpr/lpr were significantly over those in young mice. In addition, PEC chemotaxis toward FMLP concentrations did not differ between MRL-lpr/lpr and MRL-+/+ mice. Chemotactic activity of PMN FIG 2 demonstrates chemotactic activity of PMN in MRL mice. PMN obtained from MRL-+/+ mice showed increasing chemotaxis toward FMLP ranging from to 10-4M in a concentration dependent manner. The migration distances were greater in aged mice than young mice, especially significantly greater for males at all concentrations of FMLP except 10% PMN obtained from MRL-lpr/lpr mice migrated toward FMLP in a concentration dependent manner. However, migration distances did not
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SASAGAWA ET A L .
FIG 2. Chemotactic activities of PMN in MRL mice. PMN in a Boyden chamber were incubated for 90 min at 37°C under 5% COZ. The distance that PMN migrated from the surface into a 3.0 pm pore diameter Millipore fi I ter was measured. FMLP in concentrations of 10-*M to 10-4M were added t o the lower compartments of the chamber. Each point and vertical bar represents the mean and SE for 3 to 5 experiments, one of which had 3 to 4 mice. 0-- - : young males, - - - : young females, 0- : aged males, 0- : aged females.
significantly differ between young and aged mice, although those in aged mice were more than young mice. Random migrations of PMN were almost the same level between MRL-+/+ and MRL- 1 pr/ 1 pr, bet ween sexes, and be tween young and aged mice. Comparison of PMN chemotactic activities between MRL-lpr/lpr and MRL-+/+. As shown in FIG 2, chemotactic responsiveness of PMN to 10-4M of FMLP did not differ between MRL-lpr/lpr and MRL-t/t in young mice. However, in aged mice, migration distances of PMN in MRL-+/t toward 10-4M of FMLP significantly rised compared with that in MRL-lpr/lpr for both sexes. The migration distances of PMN in male MRL-t/t toward 10-5M of FMLP as well as 10-4M were significantly greater than that in male MRL- I p r / 1pr.
631
CHEMOTACTIC RESPONSE O F PMN
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DISCUSSION Studies of PMN function n murine models have commonly used PEC harvested from the peritoneal cavity. The reason is thought to be that there are some technical diff culties in isolation of PMN from small volume of murine peripheral b ood. This might make disadvantages in study ing PEC indicating that mobil zation of PMN to an nflammatory focus Metabolism and fungicidal induces marked changes in the function (19). activity of murine PEC differ from those of PMN (20). Murine PMN collected from subcutaneously implanted sponges showed significantly greater chemotactic activity than peripheral blood PMN suggesting that Rabbit peritoneal exudates showed sponge PMN are functioning PMN (21). increased chemotactic response t o FMLP in comparison with blood-borne cells (22). In the present study, first, it was found that chemotactic responsiveness to FMLP and random migration of PEC were greater than those of PMN. However, migration distance of PEC did not differ between male and female, MRL-lpr/lpr and MRL-+/+, and young and aged mice. While chemotactic activities of PMN differed between them. These results suggest that PEC have been strongly preactivated by glycogen, which is an inflammatory agent injected into the peritoneal cavity of mice in the present study. And chemotactic activities of PEC might not differ by any factors such as sex, age, and Ipr gene. Then we observed chemotactic activities of peripheral blood PMN. Chemotactic responsiveness of PMN to FMLP concentrations in young MRL-lpr/lpr were almost the same level as those in young MRL-+/+ mice. However, migration distances of PMN in aged MRL-lpr/Ipr were not so long as those in aged MRL-+/+ mice. I t is thought that PMN functions are not influenced by this disease in the early period of lifetime in mice but chemotactic responsiveness might become defective according to progress in the disease. Several abnormal i ties of PMN chemotactic responsiveness have been identified in patients with increased susceptibility to
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recurrent bacterial infections, including patients with systemic lupus erythematosus ( 1 0 , I l ) . Infection is a major cause of morbidity and It mortal i ty among patients with systemic lupus erythematosus (3,9,23). is suggested that impaired moti 1 i ty alone may be sufficient to affect host defenses adversely (24). In these types of diseases, excessively activated functions of PMN are thought to be one of major causes for development in the pathophysiology. However, it might be possible that active expression of Ipr gene could be responsive for defective chemotactic function in peripheral blood PMN. Because MRL-+/+ mice which are unaffected substrain could show increasing chemotaxis of PMN according to advancing age in the present study. MRL-lpr/lpr mice are characterized by deposition of immunocomplexes and accumulation of macrophages and histiocytic cells (12). These cells were thought to play a key role in the tissue-destruction and development of granulomatous lesions by their augmented functions such as superoxide
anion production, and enzyme release (12,251. However they were markedly decreased i n d i gest i on act i v i ty of phagocyt i zed i mmunocomp 1 exes and i n migration activity (12). PMN as well as macrophages (12) are thought to play a key role in the tissue-destruction and development of granulomat,ous lesions by their activated or depressessed functions. There are many reports demonstrating that PMN from autoimmune diseases have augmented reactive oxygen products, and lysosomal enzyme release (3-7). However, responsiveness of PMN to FMLP gradients was impaired in human rheumatoid arthritis (26) and progressive systemic sclerosis (24). Defective chemotactic responsiveness of PMN from MRL-lpr/lpr shown in the present st,udy is not contradictory to findings in human diseases (10, 11). This urges further studies to examine other functions such as releases of lysosomal enzymes and superoxide anion from PMN in MRL mice. It seems to be technically difficult to separate each type of murine leukocytes with good purity because of close densities among each type of cel Is. In the present study, centrifugation of Percoll discontinuous gradient was used for separation of PMN from peripheral blood leukocytes. This method is simple and rapid, although the purity of PMN might not be
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CHEMOTACTIC RESPONSE O F PMN
633
desirably at 100%. The obtained purity in the present study seemed to be enough to examine chemotaxis using Boyden chamber method. Because containing macrophages or monocytes cannot penetrate the f i 1 ter with pore size 3 pm and containing lymphocytes cannot migrate so fast as PMN, therefore PMN chemotaxis alone can be observed. PMN obtained by the present method seemed to reflect original functions and disease status in MRL mice. However, studies on lysosomal enzymes and superoxide anion production are essential to use highly purified PMN because of the strong effects of contaminating cells such as macrophages. In future studies, further device is needed to obtain much more purified PMN than in the present study.
REFERENCES 1. Okamoto, T., Ueda, K., Kambara, T., and Kutsuna, T., Chemotactic activity for polymorphonuclear and mononuclear leukocytes in rheumatoid synovial fluids, Acta. Pathol. Jpn., 36: 1109, 1986. 2. Palmer, D.C., Hogg, N., and Pevell, P.A., Lymphocytes, pol ymorphonuclear leukocytes, macrophages and platelets in synovium involved by rheumatoid arthritis. A study with monoclonal antibodies, Pathology, 18:431, 1986.
Bender, J. C., VanEpps, D.E., Searles, R., and Wi I 1 iams, R. C., Altered function of synvial fluid granulocytes in patients with acute inflammatory arthritis: Evidence for activation of neutrophi 1s and its mediation by a factor present in synovial fluid, Inflammation, 10:443, 3.
1986.
Blackburn, W. D., Koopman, W. J., Schrohenloher, R. E., and Heck L. W., Induction of neutrophi 1 enzyme release by rheumatoid factors: Evidence for d i fferences based on mo 1 ecu 1 ar character i st i cs, C 1 i n. Immuno1. Immunopathol., 40:347, 1986.
4.
5. Biemond, P., Swaak, A. J. C., Penders, J. M. A., Beindorff, C. M., and Kos ter, J. F. , Superox i de product i on by po 1 ymorphonuc1 ear I eucocy tes i n
rheumatoid arthritis and osteoarthritis: in vivo inhibition by the antirheumatic drug piroxicam due to interference with the activation of the NADPH-oxidase, Ann. Rheum. Dis., 45:249, 1986.
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Y., Role of stimulated neutrophi 1s from patients with systemic lupus erythematosus in tissue injury, with special reference to serum factors and increased active oxygen species generated by neutrophi Is, Inflammation, 9: 163, 1985.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Karolinska Institutet University Library on 01/26/15 For personal use only.
6. Niwa, Y., Sakane, T., Shingu, M., and Miyachi,
7. Shingu, M., Todoroki, T., Nobunaga, M., Generation of superoxide by immunologically stimulated normal numan neutrophils and possible modulation by intracel lular SOD and rheumatoid factors. Inflammation, 11 :143, 1987. 8. Dubois, E., Wierzchowiecki, M., Cos, M.B., and Weiner, J . M . , Duration and death i n systemic lupus erythematosus. An analysis of 249 cases, J. Amer. Med. Assoc., 227:1399, 1974.
9. Lee, P . , Urowitz, M. B., Bookman, A. A.M., Koehler, B. E., Smythe, H. A., Gordon, D. A . , and Ogryzlo, M. A., Systemic lupus erythematosus. A review of 110 cases with reference to nephritis, the nervous system, infections, aseptic necrosis and prognosis, Q. J. Med., 181:1, 1977.
Clark, R. A., Kimball, H. R., and Decker, J. L., Neutrophi 1 chemotaxis in systemic lupus erythematosus, Ann. Rheum. Dis., 33:167, 1974.
10.
11. Coetzl, E.J., Defective responsiveness to ascorbic acid of neutrophi 1 random and chemotactic migration in Felty’s syndrome and systemic lupus erythematosus, Ann. Rheum. Dis., 35:510, 1976.
12. Murphy, E. D., and Roths, J. B . , Autoimmunity and lymphoprol iferat ion: Induction by mutant gene Ipr, and acceleration by a male-associated factor in strain BXSB mice, I n ”Genetic control of autoimmune disease” Ed by Rose, Rigazzi, Warner, Elsevier, North Holland Inc, ~ ~ 2 0 71978. , 13. Nose, M., Tachiwaki, O., Miyazawa, M., Kanno, H., and Kyogoku, M., Macrophage functions and their regulation in MRL/I lupus mice, I n ”New Horizons in animal models for autoimmune disease” Ed by Kyogoku M., Wigzel 1 H . , Academic Press, Tokyo, Orlando, San Diego, New York, Austin, Boston, London, Sydney, Toronto, pp219, 1987. 14. Yamashita, T., Ishibashi, Y., Nagaoka, I., Kasuya, K., Masuda, K., Warabi, H., and Shiokawa, Y., Studies on glycogen-induced inflammation of mice. Inflammation, 6:87, 1982.
15. Nagaoka, I., and Yamashi ta, T., Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity, Histochemistry, 81: 273, 1984.
CHEMOTACTIC RESPONSE O F PMN
635
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16. Harbeck, R. J . , Hoffman, A.A., Redecker, S., Biundo, T., and Kurnick, J . , The isolation and functional activity of polymorphonuclear leukocytes
and lymphocytes separated from whole blood on a single Percoll density gradient, CI in. Immunol. Immunopathol., 23:682, 1982. 17. Gate, K. L., Ray, C. E., and Quie, P.G., Modified Boyden chamber method of measuring polymorphonuclear leukocyte chemotaxis, In Leukocyte Chemotaxis. Ed by Gal 1 in J. I . , and Quie P. G., New York, Raven Press, pp67, 1977. 18. Zingmond, S. H., and Hirsch, J. G., Leukocyte locomotion and chemotaxis. New methods for evaluation and demonstration of a cel I-der ived chemotactic factor, J. Exp. Med., 137:387, 1973. 19. Wanda1 I , J. H., Function of polymorphonuclear neutrophi lic leucocytes. Comparison of leukocytes from blood and exudate in heal thy volunteers, Acta. path. microbiol. scand. Sect. C., 90:7, 1982.
20. Brummer, E., McEwen, J.G., and Stevens, D . A . , Fungicidal activity of murine inflammatory polymorphonuclear neutrophi 1s: comparison with murine peripheral blood PMN, Clin. exp. Immunol., 66:681, 1986. 21. Bale, J. F., and O’NeilI, M. E., Murine neutrophils col lected from subcutaneously implanted sponges, J. Surg. Res., 39:439, 1985. 22. Kel ler, H. U., and Cotter, H., Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates, Blood Cells, 10:45, 1984.
Perez, H. D . , and Goldstein, I. M., Polymorphonuclear leukocyte chemotaxis in systemic lupus erythematosus, J. Rheumatol., 14:53, 1987 23.
24. Czirjak, L., Danko, K., Sipka, S., Zeher, M., and Szagedi, G.Y., Polymorphonuclear neutrophi 1 function in systemic sclerosis, Ann. Rheum. Dis., 46:302, 1987. 25. Rokutan, K., Hosokawa, T., Nakamura, K., Koyama, K., Aoike, A., and Kawai, K., Increased superoxide anion product ion and glutathione peroxidase activity in peritoneal macrophages from autoimmune-prone MRL/Mp-lpr/lpr mice, Int. Arch. Allergy Appl. Immunol., 87:113, 1988.
26. Turner, R. A., Johnson, J. A., and Turner, S.R., Neutrophil respons i veness to chemoatt rac tant tr ipept i de in rheumato id arthr i t is, Proc. SOC. Exp. Biol. Med., 186:125, 1987.
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 14(3), 625-635 (1992)
Chemotactic Activities of Peripheral Blood Polymorphonuclear Leukocytes and Peritoneal Exudate Polymorphonuclear Leukocytes in MRL Mice. Sumi ko Sasagawa', Yukio Satow*, Kazuo Suzuki3, Tomokazu Hosokawa4 'Radiation Effects Research Foundation, Hiroshima 732, Japan 2Hiroshima University, Hiroshima 734, Japan 3Ja~anNational Institue of Health, Tokyo 141, Japan 4Kyoto Prefectural University of Medicine, Kyoto 606, Japan
ABSTRACT Chemotactic responsiveness to fMet-Leu-Phe in concentrations of to 10-4M in the Boyden chamber was compared between peritoneal exudate cells (PEC) and polymorphonuclear leukocytes (PMN) isolated from peripheral blood, between MRL/Mp-+/+ (MRL-+/+) and MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice, and between young (6 - 9 week old) and aged (16 - 24 week old) mice. Chemotactic responsiveness of PEC did not differ between MRL-+/+ and MRL-lpr/lpr, and young and aged mice. While, PMN shored greater chemotaxis in aged MRL-+/+ mice than that in aged MRL-lpr/lpr mice. These results suggest that chemotactic responsiveness of PMN differ from that of PEC which is assumed to be preactivated by an inflammatory agent injected into the peritoneal cavity to elicit cells. Less responsiveness of PMN to the bacterial origin peptide might relate to the autoimmune disease of this murine model.
I NTRODUCTI ON Pol ymorphonuclear leukocytes (PMN) are essential for normal host defenses by virtue of their ability to seek out, recognize, ingest and ki 1 1 microorganisms. However, their excessively infIammatory response is 625 Copyright 0 1992 by Marcel Dekker, Inc.
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Karolinska Institutet University Library on 01/26/15 For personal use only.
626
SASAGAWA ET AL.
thought to be harmful. Human autoimmune diseases develop a lot of various forms of' tissue injury. In rheumatoid arthritis, a chronic state of inflammation exists i n which the synovial fluid often contains a large number of PMN (1,2). Accumulation and hyperfunction of PMN in inflammatory sites including release of lysosomal enzymes and product ion of superoxide anions are contributable to the pathogenesis of the disease (3-7). While, infection is a major cause of morbidity and mortality among patients with systemic lupus erythematosus (SLE) (8,9). Defect of PMN chemotactic responsiveness have been identified i n patients with incr eased susceptibi 1 i ty to recurrent bacterial infections,including patien ts with SLE (10, 11). MRL mice are a murine model that spontaneously develops autoimmune disease (12). MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice are we1 1 known to involve an autosomal recessive mutant genome Ipr, which produces massive T-cell proliferation and early onset of autoimmune diseases and early death with arthritis and glomerulonephritis during their second year of life (13). A I 1 MRL- I pr/ 1 pr mice deve 1op mass i ve genera I i zed 1ymph node en 1argement, etc., and early death with glomerulonephritis. MRL/Mp-t/+ (MRL-+/t) mice are an unaffected substrain which do not develop lymphoprol iferative syndrome. Their immunity and pathology have been we1 1 studied. However, PMN which are thought to play a crucial role i n the pathophysiology of the autoimmune diseases have not yet been studied in this type of mice, because of a lack of suitable methods for studying murine PMN. Human PMN are frequently isolated for studies from peripheral blood, however,the small volume of blood from mice makes this a difficult source when study i ng mur i ne PMN. Therefore many studies have used per i tonea I exudate cells (PEC) elicited by the injection of an inflammatory agent. Our preliminary trials showed that PEC of mice showed markedly enhanced superoxide anion production, suggesting that PEC had already been activated by an inflammatory procedure. In the present study, we examined migrating activity of PEC and PMN obtained from MRL-lpr/lpr and MRL-t/t mice.
CHEMOTACTIC RESPONSE OF PMN
621
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MATERIALS AND METHODS Mice : MRL-+/+ and MRL-lpr/lpr mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained at our animal facility. Six t o eight week-old mice were used as the young group and 16 to 24 week old mice as the aged group. Preparation of PEC : This procedure was done according t o the method of Yamashita et a1 (14,151 with a slight modification. PEC highly enriched for inflammatory PMN were induced by intraperitoneal injection of one ml of 15% glycogen. Six hours later, mice were killed by cervical dislocation and the peritoneal cavities were washed out. PEC were collected by lavage of the peritoneum with a 5 ml of PBS containing 5 U/ml of heparin. After removal of containing erythrocytes by a hypotonic solution of a 0.75% ammonium chloride (supplemented by 20mM Tris-HCI, PH cells were twice washed with phosphate buffered saline (PBS) by centrifugation at 400 g for 10 min at 2 O O C . 7.4),
Preparation of PMN : Heparinized (20 W m l ) peripheral blood samples obtained from three to four mice were pooled and mixed with 1.5% (W/V> dextran T-500 - PBS solution. Containing erythrocytes in the leukocyte fraction were removed by lysis with a 0.75% ammonium chloride solution containing 20 mM Tris-HCI (final pH 7.4) for 5 min at 37°C with gentle shaking. PMN enriched fraction was obtained by a centrifugation of the discontinuous density gradient of Percoll. Percoll Pharmac i a Fine Chemicals, Uppsala, Sweden) was diluted as described by Harbeck et al (16). A stock solution labelled 100% was prepared by m Xing 9 volumes of Percoll with 1 volume of 10 x PBS. The stock solut on was diluted to solutions of 65%, 60%, and 55% Percoll. Preformed gradients of 6 ml were Prepared in disposable polystyrene tubes (120 by 16 mm; Corning). Pasteur Pipettes were used to layer successively 2 ml of each of the 55, and 60% Percoll solutions onto 2 ml of the 65% solution. Finally, one ml of the leukocyte suspension (approximately 5 x lo6 cells) were loaded onto each
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628
SASAGAWA ET A L .
gradient. Tubes were centrifuged at 1600 g for 30 min at 4OC before harvest of each cell band with Pasteur pipettes. Harvested cells on the 60% Percoll laver were twice washed with PBS. Cel I counts : Differential leukocyte counts were made by examination of Wright-stained preparations of the pel leted cel Is which were smeared on a slide glass. After staining, the smeared slide glass was read under transmitted brigh field (x 40) and the proportion of granulocytic leukocytes to tot 1 leukocytes were recorded. In calculating this percentage,broken cel Is were not included. A minimum of 200 cells were counted for each smear. Percentages of granulocytic leukocytes in Wright stain were around 75 - 90% for PEC, 15 - 20% for peripheral blood leukocytes, and 65-75% for the PMN enriched fraction. These percentage values were always shown through the experiments. Measurement of migrating activity : Migration was measured using the modified Boyden chamber method (17). Cells were suspended at a concentration of 2 x 106 cells/ml in Hanks balanced salt solution (HBSS) that contained 0.9% bovine serum albumin and 0.27% NaHC03, and were incubated in a Boyden chamber (Bio-Rad Lab, Richmond, CAI for 90 min at 37°C under 5% Con. The migration distance (pm/90 m i d from the surface of a Mi 1 1 ipore f i lter (3.0 pm pore size, Mi 1 1 ipore, Bedford, MA, U. S . A. 1 The resulting t o the leading front of the cells i n it was measured (18). value was used to determine random migration and chemotaxis to fMet-Leu-Phe (FMLP).
RESULTS Chemotactic activity of PEG FIG 1 demonstrates chemotactic activity of PEC in MRL mice. PEC obtained both from MRL-lpr/lpr and MRL-+/+ mice migrated toward FMLP ranging from to 10-4M in a concentration dependent manner. The
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CHEMOTACTIC RESPONSE OF PMN
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720
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L
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I
I
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FIG 1. Chemotactic activities of PEC in MRL mice. PEC in a Boyden chamber were incubated for 90 min at 37OC under 5% C02. The distance that PEC migrated from the surface into a 3.0 pm pore diameter Millipore filter was measured. FMLP in concentrations of 10-8M to 10-4M were added to the lower compartments of the chamber. Each point and vertical bar represents the mean and SE for 6 to 8 mice. 0-- - : young males, 0 - - - : young females, 0- : aged males, 0: aged females.
chernotactic activities did not differ between male and female mice, nor between young and aged mice except at concentrations of 10-*M and 10-6M of FMLP, where chemotactic distances in aged female MRL-lpr/lpr were significantly over those in young mice. In addition, PEC chemotaxis toward FMLP concentrations did not differ between MRL-lpr/lpr and MRL-+/+ mice. Chemotactic activity of PMN FIG 2 demonstrates chemotactic activity of PMN in MRL mice. PMN obtained from MRL-+/+ mice showed increasing chemotaxis toward FMLP ranging from to 10-4M in a concentration dependent manner. The migration distances were greater in aged mice than young mice, especially significantly greater for males at all concentrations of FMLP except 10% PMN obtained from MRL-lpr/lpr mice migrated toward FMLP in a concentration dependent manner. However, migration distances did not
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SASAGAWA ET A L .
FIG 2. Chemotactic activities of PMN in MRL mice. PMN in a Boyden chamber were incubated for 90 min at 37°C under 5% COZ. The distance that PMN migrated from the surface into a 3.0 pm pore diameter Millipore fi I ter was measured. FMLP in concentrations of 10-*M to 10-4M were added t o the lower compartments of the chamber. Each point and vertical bar represents the mean and SE for 3 to 5 experiments, one of which had 3 to 4 mice. 0-- - : young males, - - - : young females, 0- : aged males, 0- : aged females.
significantly differ between young and aged mice, although those in aged mice were more than young mice. Random migrations of PMN were almost the same level between MRL-+/+ and MRL- 1 pr/ 1 pr, bet ween sexes, and be tween young and aged mice. Comparison of PMN chemotactic activities between MRL-lpr/lpr and MRL-+/+. As shown in FIG 2, chemotactic responsiveness of PMN to 10-4M of FMLP did not differ between MRL-lpr/lpr and MRL-t/t in young mice. However, in aged mice, migration distances of PMN in MRL-+/t toward 10-4M of FMLP significantly rised compared with that in MRL-lpr/lpr for both sexes. The migration distances of PMN in male MRL-t/t toward 10-5M of FMLP as well as 10-4M were significantly greater than that in male MRL- I p r / 1pr.
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CHEMOTACTIC RESPONSE O F PMN
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DISCUSSION Studies of PMN function n murine models have commonly used PEC harvested from the peritoneal cavity. The reason is thought to be that there are some technical diff culties in isolation of PMN from small volume of murine peripheral b ood. This might make disadvantages in study ing PEC indicating that mobil zation of PMN to an nflammatory focus Metabolism and fungicidal induces marked changes in the function (19). activity of murine PEC differ from those of PMN (20). Murine PMN collected from subcutaneously implanted sponges showed significantly greater chemotactic activity than peripheral blood PMN suggesting that Rabbit peritoneal exudates showed sponge PMN are functioning PMN (21). increased chemotactic response t o FMLP in comparison with blood-borne cells (22). In the present study, first, it was found that chemotactic responsiveness to FMLP and random migration of PEC were greater than those of PMN. However, migration distance of PEC did not differ between male and female, MRL-lpr/lpr and MRL-+/+, and young and aged mice. While chemotactic activities of PMN differed between them. These results suggest that PEC have been strongly preactivated by glycogen, which is an inflammatory agent injected into the peritoneal cavity of mice in the present study. And chemotactic activities of PEC might not differ by any factors such as sex, age, and Ipr gene. Then we observed chemotactic activities of peripheral blood PMN. Chemotactic responsiveness of PMN to FMLP concentrations in young MRL-lpr/lpr were almost the same level as those in young MRL-+/+ mice. However, migration distances of PMN in aged MRL-lpr/Ipr were not so long as those in aged MRL-+/+ mice. I t is thought that PMN functions are not influenced by this disease in the early period of lifetime in mice but chemotactic responsiveness might become defective according to progress in the disease. Several abnormal i ties of PMN chemotactic responsiveness have been identified in patients with increased susceptibility to
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recurrent bacterial infections, including patients with systemic lupus erythematosus ( 1 0 , I l ) . Infection is a major cause of morbidity and It mortal i ty among patients with systemic lupus erythematosus (3,9,23). is suggested that impaired moti 1 i ty alone may be sufficient to affect host defenses adversely (24). In these types of diseases, excessively activated functions of PMN are thought to be one of major causes for development in the pathophysiology. However, it might be possible that active expression of Ipr gene could be responsive for defective chemotactic function in peripheral blood PMN. Because MRL-+/+ mice which are unaffected substrain could show increasing chemotaxis of PMN according to advancing age in the present study. MRL-lpr/lpr mice are characterized by deposition of immunocomplexes and accumulation of macrophages and histiocytic cells (12). These cells were thought to play a key role in the tissue-destruction and development of granulomatous lesions by their augmented functions such as superoxide
anion production, and enzyme release (12,251. However they were markedly decreased i n d i gest i on act i v i ty of phagocyt i zed i mmunocomp 1 exes and i n migration activity (12). PMN as well as macrophages (12) are thought to play a key role in the tissue-destruction and development of granulomat,ous lesions by their activated or depressessed functions. There are many reports demonstrating that PMN from autoimmune diseases have augmented reactive oxygen products, and lysosomal enzyme release (3-7). However, responsiveness of PMN to FMLP gradients was impaired in human rheumatoid arthritis (26) and progressive systemic sclerosis (24). Defective chemotactic responsiveness of PMN from MRL-lpr/lpr shown in the present st,udy is not contradictory to findings in human diseases (10, 11). This urges further studies to examine other functions such as releases of lysosomal enzymes and superoxide anion from PMN in MRL mice. It seems to be technically difficult to separate each type of murine leukocytes with good purity because of close densities among each type of cel Is. In the present study, centrifugation of Percoll discontinuous gradient was used for separation of PMN from peripheral blood leukocytes. This method is simple and rapid, although the purity of PMN might not be
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desirably at 100%. The obtained purity in the present study seemed to be enough to examine chemotaxis using Boyden chamber method. Because containing macrophages or monocytes cannot penetrate the f i 1 ter with pore size 3 pm and containing lymphocytes cannot migrate so fast as PMN, therefore PMN chemotaxis alone can be observed. PMN obtained by the present method seemed to reflect original functions and disease status in MRL mice. However, studies on lysosomal enzymes and superoxide anion production are essential to use highly purified PMN because of the strong effects of contaminating cells such as macrophages. In future studies, further device is needed to obtain much more purified PMN than in the present study.
REFERENCES 1. Okamoto, T., Ueda, K., Kambara, T., and Kutsuna, T., Chemotactic activity for polymorphonuclear and mononuclear leukocytes in rheumatoid synovial fluids, Acta. Pathol. Jpn., 36: 1109, 1986. 2. Palmer, D.C., Hogg, N., and Pevell, P.A., Lymphocytes, pol ymorphonuclear leukocytes, macrophages and platelets in synovium involved by rheumatoid arthritis. A study with monoclonal antibodies, Pathology, 18:431, 1986.
Bender, J. C., VanEpps, D.E., Searles, R., and Wi I 1 iams, R. C., Altered function of synvial fluid granulocytes in patients with acute inflammatory arthritis: Evidence for activation of neutrophi 1s and its mediation by a factor present in synovial fluid, Inflammation, 10:443, 3.
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Blackburn, W. D., Koopman, W. J., Schrohenloher, R. E., and Heck L. W., Induction of neutrophi 1 enzyme release by rheumatoid factors: Evidence for d i fferences based on mo 1 ecu 1 ar character i st i cs, C 1 i n. Immuno1. Immunopathol., 40:347, 1986.
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5. Biemond, P., Swaak, A. J. C., Penders, J. M. A., Beindorff, C. M., and Kos ter, J. F. , Superox i de product i on by po 1 ymorphonuc1 ear I eucocy tes i n
rheumatoid arthritis and osteoarthritis: in vivo inhibition by the antirheumatic drug piroxicam due to interference with the activation of the NADPH-oxidase, Ann. Rheum. Dis., 45:249, 1986.
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Y., Role of stimulated neutrophi 1s from patients with systemic lupus erythematosus in tissue injury, with special reference to serum factors and increased active oxygen species generated by neutrophi Is, Inflammation, 9: 163, 1985.
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6. Niwa, Y., Sakane, T., Shingu, M., and Miyachi,
7. Shingu, M., Todoroki, T., Nobunaga, M., Generation of superoxide by immunologically stimulated normal numan neutrophils and possible modulation by intracel lular SOD and rheumatoid factors. Inflammation, 11 :143, 1987. 8. Dubois, E., Wierzchowiecki, M., Cos, M.B., and Weiner, J . M . , Duration and death i n systemic lupus erythematosus. An analysis of 249 cases, J. Amer. Med. Assoc., 227:1399, 1974.
9. Lee, P . , Urowitz, M. B., Bookman, A. A.M., Koehler, B. E., Smythe, H. A., Gordon, D. A . , and Ogryzlo, M. A., Systemic lupus erythematosus. A review of 110 cases with reference to nephritis, the nervous system, infections, aseptic necrosis and prognosis, Q. J. Med., 181:1, 1977.
Clark, R. A., Kimball, H. R., and Decker, J. L., Neutrophi 1 chemotaxis in systemic lupus erythematosus, Ann. Rheum. Dis., 33:167, 1974.
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11. Coetzl, E.J., Defective responsiveness to ascorbic acid of neutrophi 1 random and chemotactic migration in Felty’s syndrome and systemic lupus erythematosus, Ann. Rheum. Dis., 35:510, 1976.
12. Murphy, E. D., and Roths, J. B . , Autoimmunity and lymphoprol iferat ion: Induction by mutant gene Ipr, and acceleration by a male-associated factor in strain BXSB mice, I n ”Genetic control of autoimmune disease” Ed by Rose, Rigazzi, Warner, Elsevier, North Holland Inc, ~ ~ 2 0 71978. , 13. Nose, M., Tachiwaki, O., Miyazawa, M., Kanno, H., and Kyogoku, M., Macrophage functions and their regulation in MRL/I lupus mice, I n ”New Horizons in animal models for autoimmune disease” Ed by Kyogoku M., Wigzel 1 H . , Academic Press, Tokyo, Orlando, San Diego, New York, Austin, Boston, London, Sydney, Toronto, pp219, 1987. 14. Yamashita, T., Ishibashi, Y., Nagaoka, I., Kasuya, K., Masuda, K., Warabi, H., and Shiokawa, Y., Studies on glycogen-induced inflammation of mice. Inflammation, 6:87, 1982.
15. Nagaoka, I., and Yamashi ta, T., Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity, Histochemistry, 81: 273, 1984.
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16. Harbeck, R. J . , Hoffman, A.A., Redecker, S., Biundo, T., and Kurnick, J . , The isolation and functional activity of polymorphonuclear leukocytes
and lymphocytes separated from whole blood on a single Percoll density gradient, CI in. Immunol. Immunopathol., 23:682, 1982. 17. Gate, K. L., Ray, C. E., and Quie, P.G., Modified Boyden chamber method of measuring polymorphonuclear leukocyte chemotaxis, In Leukocyte Chemotaxis. Ed by Gal 1 in J. I . , and Quie P. G., New York, Raven Press, pp67, 1977. 18. Zingmond, S. H., and Hirsch, J. G., Leukocyte locomotion and chemotaxis. New methods for evaluation and demonstration of a cel I-der ived chemotactic factor, J. Exp. Med., 137:387, 1973. 19. Wanda1 I , J. H., Function of polymorphonuclear neutrophi lic leucocytes. Comparison of leukocytes from blood and exudate in heal thy volunteers, Acta. path. microbiol. scand. Sect. C., 90:7, 1982.
20. Brummer, E., McEwen, J.G., and Stevens, D . A . , Fungicidal activity of murine inflammatory polymorphonuclear neutrophi 1s: comparison with murine peripheral blood PMN, Clin. exp. Immunol., 66:681, 1986. 21. Bale, J. F., and O’NeilI, M. E., Murine neutrophils col lected from subcutaneously implanted sponges, J. Surg. Res., 39:439, 1985. 22. Kel ler, H. U., and Cotter, H., Comparison of locomotion, chemotaxis and adhesiveness of rabbit neutrophils from blood and peritoneal exudates, Blood Cells, 10:45, 1984.
Perez, H. D . , and Goldstein, I. M., Polymorphonuclear leukocyte chemotaxis in systemic lupus erythematosus, J. Rheumatol., 14:53, 1987 23.
24. Czirjak, L., Danko, K., Sipka, S., Zeher, M., and Szagedi, G.Y., Polymorphonuclear neutrophi 1 function in systemic sclerosis, Ann. Rheum. Dis., 46:302, 1987. 25. Rokutan, K., Hosokawa, T., Nakamura, K., Koyama, K., Aoike, A., and Kawai, K., Increased superoxide anion product ion and glutathione peroxidase activity in peritoneal macrophages from autoimmune-prone MRL/Mp-lpr/lpr mice, Int. Arch. Allergy Appl. Immunol., 87:113, 1988.
26. Turner, R. A., Johnson, J. A., and Turner, S.R., Neutrophil respons i veness to chemoatt rac tant tr ipept i de in rheumato id arthr i t is, Proc. SOC. Exp. Biol. Med., 186:125, 1987.
