Endothelial Cell Destruction by Polymorphonuclear Leukocytes Incubated with Sera from Patients w'rth Systemic Lupus Erythematosus (SLE) Y. Hashimoto, K. Nakano, S. Yoshinoya, K. Tanimoto and K. ltoh Department of internal Medicine & Physical Therapy, University of Tokyo School of Medicine, Tokyo, Japan
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Hashimoto Y, Nakano K, Yoshinoya S, Tanimoto K, Itoh K. Endothelial Cell Destruction by Polymorphonuclear Leukocytes Incubated with Sera from Patients with Systemic Lupus Erythematosus (SLE). Scand J Rheumatol 1992; 21: 209-214. When normal polymorphonuclear leukocytes (PMN) were incubated with sera from patients with active systemic lupus erythematows (SLE), a significantly increased cytotoxicity against human cultured vascular endothelial cells (EC), compared with normal control sera, was demonstrated by the standard 5'Cr release method. The degree of this cytotoxicity was correlated with the immune complex level in each serum. The cytotoxicity did not correlate with the presence of anti-EC antibody. An absorption study with Clq-Sepharose 4B further suggested that the immune complexes are the factor which induce cytotoxicity. A gel fractionation study, however, indicated the heterogenity of the cytotoxic activity, and suggested the possible contribution of other substances including anti-EC, at least in some of the patients. This type of cytotoxicity may initiate the inflammatory process including vascular damage of the disease.
Key words: systemic lupus erythematosus, polyrnorphonuclear leukocytes, endothelial cells, immune complexes, anti-endothelial cell antibody.
Systemic lupus erythematosus (SLE), a chronic inflammatory disease involving multi-organ systems, is characterized by the appearance of a variety of autoantibodies. Although the precise mechanism of autoantibody production remains to be elucidated, it is widely recognized that these antibodies are closely related to the tissue damage in SLE, either in a direct manner (autoimmune hemolytic anemia), or via the formation of immune complexes. Immune complexes, which may be the products of an abberated immune circuit of the host, might therefore initiate the inflammatory process of the disease. Experimental serum sickness is a model of immune complex-mediated tissue damage, and the contribution of polymorphonuclear leukocytes (PMN) in the disease process has been extensively investigated (1). On the other hand, it has been reported (2-5) that approximately one-third of sera from the patients with SLE contains anti-endothelial cell antibody and that its presence may be related to vasculitic lesions (2).
Yoshimi Hashimoto, Department of Internal Medicine & Physical Therapy, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113 Japan Received 30 September 1991 Accepted 16 May 1992
Systemic necrotizing vasculitis is one of the characteristic features of connective tissue diseases including SLE. Based on these viewpoints, it is beneficial to study the possible contribution of PMN in the development of vasculitic injury in SLE. In the present study, we examined whether normal human PMN, when incubated with the sera from SLE patients, acquire cytotoxicity against human vascular endothelial cells (EC) and whether this cytotoxicity is related to the presence of immune complexes or anti-EC antibody.
Material and Methods Patients Serum samples were obtained from 24 patients with SLE (2 males, 22 females; mean age 34) and 18 healthy subjects (2 males and 16 females; mean age 36). All SLE patients satisfied the 1982 Revised American Rheumatism Association Classification Criteria for SLE. Patients were divided into two groups: clinically active (18 patients) and inactive (6 patients). This was performed according to the guidelines previously described (6) with a slight modification. Patients with active disease had either one or more organ systems involved and fever, or involvement of two or more organ systems but not fever. Patients with inactive SLE had no clinical evidence of disease activity. Five patients with active disease had clinically obvious 209
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Y . Hashimoto et al. skin vasculitis (such as ulcer, urticaria or purpura). No patient was receiving more than 30 mg daily of prednisolone or cytotoxic drugs.
gammacounter. The assay was done in triplicate and the percent W r release was calculated from the following formula;
Human polymorphonuclear leukocytes (PMN) PMN were separated from heparinized venous blood from normal healthy donors by the standard method of Ficoll-Hypaque gradient centrifugation, dextran sedimentation, and hypotonic lysis of red blood cells as previously described (7). PMN were suspended in Hanks' balanced salt solution (HBSS) for further experiments.
percent "Cr release = (A-C)/(B-C) x 100%
Endothelial cells (EC) A slight modification of the technique of Jaffe et a1 (8) was employed for the culture of human EC. The lumen of a fresh umbilical cord was washed extensively with phosphate buffered saline (PBS) and digested with 0.2% collagenase in PBS for 15 min at 37°C. The collagenase solution was then flushed from the cords and washed twice with PBS. All the effluent was collected, the cells were washed twice and suspended in culture medium. The culture medium consisted of Medium-199 supplemented with 20% fetal calf serum (FCS), penicilline (200 unitdml), streptomycin (100 pg/ml), L-glutamine (2 mM), heparin (90 pg/ml), and endothelial cell growth supplement (20 pg/ml, Collaborative Res. Inc., Bedford MA). EC were cultured in a 25 cm2 flask, and fed every third day; they generally became confluent in 3 to 5 days. Identification of E C was confirmed by the characteristic growth pattern by phase contrast microscopy and also by the detection of factor VIII antigen by indirect immunofluorescence. Confluent EC were harvested by treatment with a mixture of 0.05% trypsin and 0.01YO EDTA and subcultured at 1:3 split; the experiments were performed using 3 to 5 passaged cells.
The C l q solid phase assay was performed as reported previously (1). Briefly, polystyrene tubes were coated with 0.5 ml purified human C l q (5 pg/ml) for 16 h at 4°C. Remaining binding sites were blocked with 1% bovine serum albumin (BSA). Sample sera (0.005 ml) were added to the tubes, and BSA solution was added to bring the final volume to 0.5 ml. After 16 h at 4°C the tubes were washed three times and the immune complexes bound to C l q were measured using labeled Fab rabbit antiserum specific for human Fab. The results were recorded as the amount of heataggregated IgG equivalent as reported previously (10).
Cytotoxicity assay This was performed as previously described (9). Briefly, EC were suspended at 7.5x105/ml in Medium-199 with 5% FCS to which 'lCr was added to make a concentration of 5 pCi/ml. The 200 pl of cell suspension was distributed to each well of Falcon Microtest I1 and left overnight at 37°C to allow the cells to attach to the plate and take up 51Cr. The cells were washed five times with warm HBSS and served for the assay. The medium was replaced with 100 pl of 40% test serum in Medium-199. The assay was initiated by adding 100 pl of PMN solution (7.5 X lo4 cells) to achieve an effectodtarget ratio of 5. After 5 hours of incubation at 37°C with 5% C 0 2 and 100% humidity, the plate was centrifuged at 100 g for 5 min. Each 100 pl of supernatant was collected and counted in a 210
where A is the mean cpm in the experimental supernatant, B is the mean cpm of total release obtained by Triton-X (0.2%) treatment of EC, and C is the spontaneous release.
Immune complexes
Absorption of immune complexes The Clq-Sepharose 4B was prepared from the Sepharose 4B (50 ml) activated with cyanogen bromide. It was conjugated overnight with the purified C l q solution (2 mg/ml X 2.5 ml). The gel was washed extensively with PBS and blocked with 1M glycine to eliminate the remaining protein binding sites. The final product was taken up into a 50 ml syringe, washed with PBS until the eluate indicated zero reading at OD 280 nm. As 100% of C l q was thought to bind to Sepharose 4B, the gel contained 0.1 mg of C l q per 1 ml of Sepharose 4B suspension. One milliliter of the serum sample was conjugated with 1.5 ml of Clq-Sepharose 4B at 4°C for 4 hours. The mixture was centrifuged at 800 rpm for 20 minutes and the supernate was carefully moved into a new tube. The supernate was then extensively dialyzed against M-199 medium and served for the assay. For control, the serum was treated in the same manner with the Clq-Sepharose 4B not conjugated with Clq, but blocked with glycine. Anti-EC antibody Anti-EC antibody analysis was performed according to a method previously reported (4) with a slight modification. Briefly, E C were seeded at 2 x 104/well in Falcon 96-well flat bottom microplate coated with 1% gelatin and cultured in M-199 me-
P M N Mediated Endothelial Damage in SLE specific release with PMN in 20% FCS medium was 1.2 f 1.5 (SD) YO.When PMN were incubated in the presence of normal sera, the mean cytotoxicity was 1.5 f 2.0 (SD) %, whereas it was 7.8 f 5.5% in the case of active SLE sera and 1.1 f 1.1% with inactive SLE. The difference between normal control and active SLE group was statistically significant (p < 0.01, Student's t-test). Four out of five sera with dermal vasculitis showed the raised cytotoxicity. SLE sera alone without PMN, as well as normal sera, were not cytotoxic during the 5-hours assay (data not shown).
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Fig. 1. Summary of the EC cytotoxicity assay mediated by PMN incubated with normal or SLE sera (active and inactive). Em ratio was 5 and incubation time was 5 hours. The horizontal line in the left column represents the value of the mean plus two standard deviations with normal sera. The difference between normal and active SLE groups was statistically significant (p < 0.01, Student's t-test). Filled circles indicate the values from sera from patients with skin vasculitis.
dium with 10% FCS for 24 hours. The medium was replaced with test serum 25 times diluted with HBSS in triplicate. After one hour at 37"C, sera were aspirated and EC were washed 3 times with HBSS. One hundred microliters of peroxidaseconjugated goat anti-human IgG, diluted at 1:4000, was added and incubated for one hour at room temperature. After washing 3 times with HBSS, 100 p1 peroxidase substrate was added to each well and incubated for 15 min. The reaction was terminated with 3 M H,S04 and the OD at 492 nm was measured by an ELISA reader. All 18 normal sera and 24 SLE sera were tested on the same day employing the same source of EC.
Gel fractionation of serum The serum samples were fractionated by gel filtration on a Sephadex G-200 column (2.7 X 100 cm) equilibrated with PBS at 4°C. One milliliter of serum was passed over the column and the fractions were collected at 5 ml/tube. Fractions for three major peaks and between the peaks were concentrated to the original volume, dialyzed against M-199 and applied to the cytotoxicity assay.
Correlation of the cytotoxicity with serum C3 levels and the circulating immune complexes Figure 2 demonstrates the correlation between the serum immune complex levels and the E C cytotoxicity-inducing activities of SLE sera. A positive significant correlation (p < 0.05) was detected between the two parameters. No significant correlation was detected between the C3 levels and activities (data not shown). Absorption of serum Two sera which showed high activity and contained a relatively large amount of immune complexes were incubated with Sepharose 4B conjugated and not conjugated with Clq. Both the original and treated sera were assessed simultaneously in the cytotoxicity assay. The dilution rates of the sera were standardized by the concentrations of albumin. As demonstrated in Fig. 3, in one serum, the E C cytotoxicity-inducing activity was decreased by 86.5% when treated with Clq-Sepharose 4B and
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Effect of sera on PMN-mediated cytotoxicity The results are demonstrated in Fig. 1. Spontaneous 51Crrelease (EC in M-199 with 20% FCS) was between 7 and 10% in the 5-hour assay and the 211
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before after Fig. 3. Serum absorption with Sepharose 4B. EC cytotoxicityinducing activity of the serum compared before and after absorption of the serum with conjugated (solid lines) Sepharose 4B and not conjugated (dotted lines) Sepharose 4B with C l q .
36.5% with non-conjugated Sepharose 4B. In another serum, the results were 64.0% and 20.2%, respectively.
Correlation with anti-EC antibody Since all the sera were tested under identical conditions, values greater than mean plus 2 standard deviations of the 18 normal sera were considered as anti-EC positive. Thus, 8 out of 24 SLE sera were judged anti-EC positive and the others were negative. Figure 4 shows the relation between the presence of anti-EC antibody and the E C cytotoxicity-inducing activity. No significant difference was detected between the two groups. However, 5 out of 8 (62.5%) anti-EC posititive sera elicited an increased EC cytotoxicity-inducing activity, while 31.3% with anti-EC negative sera did. Of the five patients with dermal vasculitis, 3 were positive and the others were negative. Gel filtration of sera The three SLE sera which showed a high activity were gel filtrated on a Sephadex G-200 gel column and fractionated. Six fractions were chosen and tested for the ability to induce cytotoxicity in the assay. A normal serum was treated and applied to the assay simultaneously. In Fig. 5a-c, the results of three SLE sera are demonstrated. In the first SLE case (Fig. 5a), major activity seemed to exist in both the first and second peaks. In the second SLE case (Fig. 5b), activity mainly existed in the first peak and for the third case (Fig. 5c), in the second peak. The first and third cases were anti-EC positive and second case was negative. In contrast, no activity was detected in any fraction from the normal serum (data not shown).
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Discussion
It is well documented that polymorphonuclear leukocytes (PMN) are capable of destroying mammalian cultured cells of either tumor or non-tumor origin without accompanying phagocytosis, that is, extracellular lysis, when they are stimulated with various ligands (11, 12). It is also reported that PMN are able to destroy target cells through a specific antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism (13, 14). Endothelial cells (EC) destruction by PMN has also been demonstrated by several authors including us (9, 15, 16). The substances which stimulate PMN and initiate the cytotoxic response include chemotactic factors (15), chemical agents (16) and phagocytic stimuli including immune complexes (9). It has been suggested that this cytotoxicity may play a critical role in the immune complexmediated tissue injury, especially vasculitis. In the previous study (17), we reported that when human PMN are incubated with SLE sera they generate an elevated amount of superoxide anion and adhere more abundantly to vascular EC. We concluded that these phenomenon are ascribed to the presence of immune complexes and this we confirmed in another study (18). In the present study, we demonstrated that normal PMN, when incubated with some active SLE sera, acquired cytotoxicity against human cultured vascular EC. Inactive SLE sera as well as normal control sera did not elicit such activity. Furthermore, SLE sera in itself did not possess cytotoxic activity during the 5-hour assay. Although the degree of 51Crre-
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Anti-EC (t) Anti-EC (-1 (n= 8 ) (n=16) Fig. 4. Comparison of the cytotoxicity-inducing activity between anti-EC positive and negative SLE sera. The difference between the two groups was statistically not significant. The horizontal line indicates the normal range. Filled circles indicate the patients with skin vasculitis.
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Fig. 5a-c. Sera from 3 SLE patients were gel fractionated over a Sephadex G-200 column. Six fractions were chosen, dialyzed, concentrated to the original volume and applied to the cytotoxicity assay. Fig. 5a,b,c represent the results of 3 patients where patients a and c were anti-EC positive and b was negative. The curved lines indicate the elution patterns monitored at OD 280 nm. Bars represent the values in the cytotoxicity assay performed using each fraction for PMN stimulation.
lease was not remarkable, we considered it to be significant as also emphasized by others (15). In consideration of possible PMN stimulatory substances in SLE sera, the correlation between this activity and either serum immune complex levels or the presence of anti-EC antibody was studied. A significant correlation was observed between the activity and the immune complex level. Furthermore, incubation of the active sera with Clq-Sepharose 4B remarkably reduced the cytotoxicity, further suggesting a contribution of circulating immune complexes in the cytotoxic activity.
The important role of immune complexes and PMN in the pathogenesis of serum sickness and so called leukocytoclastic vasculitis has been emphasized. Therefore, we speculate that this type of tissue injury mechanism may actually take place in SLE. A reduction in the activity was also observed, to a much lesser degree, in the sera treated with control Sepharose beads. We suppose that this is merely a result of the nonspecific absorption. On the other hand, no significant correlation between the activity and the presence of anti-EC was demonstrated, although anti-EC positive sera tended to induce the increased activity compared with negative sera as shown in Fig. 4.The presence of anti-EC antibody in SLE was first suggested by Shingu & Hurd (19) and was established in subsequent studies (2-5). Some of these studies showed that the presence of anti-EC antibodies correlate with vascular injury. In an animal study, furthermore, it was reported that anti-EC antibody initiates the immune complex-type glomerulonephritis (20). The results of the gel fractionation study indicated that the activity under discussion may be heterogeneous and may vary among patients. Therefore, we could not deny the possible contribution of the anti-EC antibody to cytotoxicity. In our study, it was demonstrated that the majority of the sera with dermal vasculitis contained this activity. However, it is necessary to be cadtious when discussing the relationship between the results of in vitro experiments and the clinical skin vasculitis in SLE, since it has been suggested that many other deep organ involvements, for example, CNS lupus or lupus retinopathy, are also closely related with vasculitis. In conclusion, it can be suggested that this type of E C cytotoxicity may, at least in part, contribute to the initiation of the inflammatory process of SLE and the cytotoxic activity may be mediated by circulating immune complexes and presumably by other mechanisms including anti-EC. Acknowledgement We thank Miss Kyoko Shimada for her excellent technical assistance. We also wish to thank the staff of the Tokyo Metropolitan Tsukiji Obstetric Hospital for providing umbilical cords. Supported by Manabe Medical Foundation.
References 1. Kniker WT, Cochrane CG. Pathogenetic factors in vascular lesions of experimental serum sickness. J Exp Med 1965; 122: 83-98. 2. Cines DB, Lyss AP, Reeber M, Bina M, DeHoratius RJ. Presence of complement fixing anti-endothelial cell anti-
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Y. Hashimoto et al. bodies in systemic lupus erythematosus. J Clin Invest
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1984; 73: 611-25. 3. LeRoux G, Wautier MP, Guillevin L, Wautier JL. IgG
12. Weiss SJ, LoBuglio AF. An oxygen dependent mechanism of neutrophil-mediated cytotoxicity. Blood 1980; 55:
10204.
binding to endothelial cells in systemic lupus erythematosus. Thromb Haemost 1986; 56: 144-50. 4. Hashemi S, Douglas-Smith C, Izaguirre CA. Anti-endothelial cell antibodies: Detection and characterization using a cellular enzyme-linked immunosorbent assay. J Lab Clin Med 1987; 109: 434-40. 5. Vismara A, Meroni PL, Tincani A, et al. Relationship between anti-cardiolipin and anti-endothelial cell antibodies in systemic lupus erythematosus. Clin Exp Immunol
13. Gale RP, Zighelboim J. Modulation of polymorphonu-
1988; 74: 247-53. 6. Rothfield NF. Pace N. Relation of positive LE-cell preparations to activity of lupus erythematosus and corticosteroid therapy. N Engl J Med 1962; 266: 535-8. 7. Boyum A. Isolation of monouclear cells and granulocytes from human blood. J Lab Clin Med 1968; 21: 77-89. 8. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-56. 9. Hashimoto Y, Tanimoto K, Miyamoto T. Destruction of red blood cells and cultured vascular endothelial cells by immune activated polymorphonuclear leukocytes. Inflammation 1987; 11: 201-10. 10. Yoshinoya S, McDaffy S, Alarcon-Segovia D, Pope RM. Detection and partial characterization of immune complex in patients with rheumatoid arthritis plus Sjagren syndrome alone. Clin Exp lmmunol 1982; 48: 339-47. 11. Clark AR, Klebanoff SJ. Neutrophil-mediated tumor cell cytotoxicity: role of the peroxidase system. J Exp Med 1975: 141: 1442-7.
61: 1161-7. 16. Weiss SJ, Young J, LoBuglio AF, Slivka A. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J Clin Invest 1981; 68: 714-21. 17. Hashimoto Y, Ziff M, Hurd ER. Increased endothelial
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clear leukocytes-mediated antibody-dependent cellular cytotoxicity. J Immunol 1974; 113: 1793-800. 14. Clark AR, Klebanoff SJ. Studies on the mechanism of antibody-dependent polymorphonuclear leukocyte-mediated cytotoxicity. J Immunol 1977; 119: 1413-8. 15. Sachs T , Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. J Clin Invest 1978;
cell adherence, aggregation, and superoxide generation by neutrophils incubated in systemic lupus erythematosus and Felty’s syndrome sera. Arthritis Rheum 1982; 25: 1409-18. 18. Hashimoto Y, Hurd ER. Human neutrophil aggregation and increased adherence to human endothelial cells induced by heat aggregated IgG and immune complexes. Clin Exp Immunol 1981; 44: 53847. 19. Shingu M, Hurd ER. Sera from patients with systemic lupus erythematosus reactive with human endothelial cells. J Rheumatol. 1981; 8: 581-6. 20. Matsuo S, Fukatsu A , Taub ML, Caldwell PRB, Brentjens JR, Andres G . Glomerulonephritis induced in the rabbit by anti-endothelial antibody. J Clin Invest 1987; 79: 1798811.
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Hashimoto Y, Nakano K, Yoshinoya S, Tanimoto K, Itoh K. Endothelial Cell Destruction by Polymorphonuclear Leukocytes Incubated with Sera from Patients with Systemic Lupus Erythematosus (SLE). Scand J Rheumatol 1992; 21: 209-214. When normal polymorphonuclear leukocytes (PMN) were incubated with sera from patients with active systemic lupus erythematows (SLE), a significantly increased cytotoxicity against human cultured vascular endothelial cells (EC), compared with normal control sera, was demonstrated by the standard 5'Cr release method. The degree of this cytotoxicity was correlated with the immune complex level in each serum. The cytotoxicity did not correlate with the presence of anti-EC antibody. An absorption study with Clq-Sepharose 4B further suggested that the immune complexes are the factor which induce cytotoxicity. A gel fractionation study, however, indicated the heterogenity of the cytotoxic activity, and suggested the possible contribution of other substances including anti-EC, at least in some of the patients. This type of cytotoxicity may initiate the inflammatory process including vascular damage of the disease.
Key words: systemic lupus erythematosus, polyrnorphonuclear leukocytes, endothelial cells, immune complexes, anti-endothelial cell antibody.
Systemic lupus erythematosus (SLE), a chronic inflammatory disease involving multi-organ systems, is characterized by the appearance of a variety of autoantibodies. Although the precise mechanism of autoantibody production remains to be elucidated, it is widely recognized that these antibodies are closely related to the tissue damage in SLE, either in a direct manner (autoimmune hemolytic anemia), or via the formation of immune complexes. Immune complexes, which may be the products of an abberated immune circuit of the host, might therefore initiate the inflammatory process of the disease. Experimental serum sickness is a model of immune complex-mediated tissue damage, and the contribution of polymorphonuclear leukocytes (PMN) in the disease process has been extensively investigated (1). On the other hand, it has been reported (2-5) that approximately one-third of sera from the patients with SLE contains anti-endothelial cell antibody and that its presence may be related to vasculitic lesions (2).
Yoshimi Hashimoto, Department of Internal Medicine & Physical Therapy, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113 Japan Received 30 September 1991 Accepted 16 May 1992
Systemic necrotizing vasculitis is one of the characteristic features of connective tissue diseases including SLE. Based on these viewpoints, it is beneficial to study the possible contribution of PMN in the development of vasculitic injury in SLE. In the present study, we examined whether normal human PMN, when incubated with the sera from SLE patients, acquire cytotoxicity against human vascular endothelial cells (EC) and whether this cytotoxicity is related to the presence of immune complexes or anti-EC antibody.
Material and Methods Patients Serum samples were obtained from 24 patients with SLE (2 males, 22 females; mean age 34) and 18 healthy subjects (2 males and 16 females; mean age 36). All SLE patients satisfied the 1982 Revised American Rheumatism Association Classification Criteria for SLE. Patients were divided into two groups: clinically active (18 patients) and inactive (6 patients). This was performed according to the guidelines previously described (6) with a slight modification. Patients with active disease had either one or more organ systems involved and fever, or involvement of two or more organ systems but not fever. Patients with inactive SLE had no clinical evidence of disease activity. Five patients with active disease had clinically obvious 209
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Y . Hashimoto et al. skin vasculitis (such as ulcer, urticaria or purpura). No patient was receiving more than 30 mg daily of prednisolone or cytotoxic drugs.
gammacounter. The assay was done in triplicate and the percent W r release was calculated from the following formula;
Human polymorphonuclear leukocytes (PMN) PMN were separated from heparinized venous blood from normal healthy donors by the standard method of Ficoll-Hypaque gradient centrifugation, dextran sedimentation, and hypotonic lysis of red blood cells as previously described (7). PMN were suspended in Hanks' balanced salt solution (HBSS) for further experiments.
percent "Cr release = (A-C)/(B-C) x 100%
Endothelial cells (EC) A slight modification of the technique of Jaffe et a1 (8) was employed for the culture of human EC. The lumen of a fresh umbilical cord was washed extensively with phosphate buffered saline (PBS) and digested with 0.2% collagenase in PBS for 15 min at 37°C. The collagenase solution was then flushed from the cords and washed twice with PBS. All the effluent was collected, the cells were washed twice and suspended in culture medium. The culture medium consisted of Medium-199 supplemented with 20% fetal calf serum (FCS), penicilline (200 unitdml), streptomycin (100 pg/ml), L-glutamine (2 mM), heparin (90 pg/ml), and endothelial cell growth supplement (20 pg/ml, Collaborative Res. Inc., Bedford MA). EC were cultured in a 25 cm2 flask, and fed every third day; they generally became confluent in 3 to 5 days. Identification of E C was confirmed by the characteristic growth pattern by phase contrast microscopy and also by the detection of factor VIII antigen by indirect immunofluorescence. Confluent EC were harvested by treatment with a mixture of 0.05% trypsin and 0.01YO EDTA and subcultured at 1:3 split; the experiments were performed using 3 to 5 passaged cells.
The C l q solid phase assay was performed as reported previously (1). Briefly, polystyrene tubes were coated with 0.5 ml purified human C l q (5 pg/ml) for 16 h at 4°C. Remaining binding sites were blocked with 1% bovine serum albumin (BSA). Sample sera (0.005 ml) were added to the tubes, and BSA solution was added to bring the final volume to 0.5 ml. After 16 h at 4°C the tubes were washed three times and the immune complexes bound to C l q were measured using labeled Fab rabbit antiserum specific for human Fab. The results were recorded as the amount of heataggregated IgG equivalent as reported previously (10).
Cytotoxicity assay This was performed as previously described (9). Briefly, EC were suspended at 7.5x105/ml in Medium-199 with 5% FCS to which 'lCr was added to make a concentration of 5 pCi/ml. The 200 pl of cell suspension was distributed to each well of Falcon Microtest I1 and left overnight at 37°C to allow the cells to attach to the plate and take up 51Cr. The cells were washed five times with warm HBSS and served for the assay. The medium was replaced with 100 pl of 40% test serum in Medium-199. The assay was initiated by adding 100 pl of PMN solution (7.5 X lo4 cells) to achieve an effectodtarget ratio of 5. After 5 hours of incubation at 37°C with 5% C 0 2 and 100% humidity, the plate was centrifuged at 100 g for 5 min. Each 100 pl of supernatant was collected and counted in a 210
where A is the mean cpm in the experimental supernatant, B is the mean cpm of total release obtained by Triton-X (0.2%) treatment of EC, and C is the spontaneous release.
Immune complexes
Absorption of immune complexes The Clq-Sepharose 4B was prepared from the Sepharose 4B (50 ml) activated with cyanogen bromide. It was conjugated overnight with the purified C l q solution (2 mg/ml X 2.5 ml). The gel was washed extensively with PBS and blocked with 1M glycine to eliminate the remaining protein binding sites. The final product was taken up into a 50 ml syringe, washed with PBS until the eluate indicated zero reading at OD 280 nm. As 100% of C l q was thought to bind to Sepharose 4B, the gel contained 0.1 mg of C l q per 1 ml of Sepharose 4B suspension. One milliliter of the serum sample was conjugated with 1.5 ml of Clq-Sepharose 4B at 4°C for 4 hours. The mixture was centrifuged at 800 rpm for 20 minutes and the supernate was carefully moved into a new tube. The supernate was then extensively dialyzed against M-199 medium and served for the assay. For control, the serum was treated in the same manner with the Clq-Sepharose 4B not conjugated with Clq, but blocked with glycine. Anti-EC antibody Anti-EC antibody analysis was performed according to a method previously reported (4) with a slight modification. Briefly, E C were seeded at 2 x 104/well in Falcon 96-well flat bottom microplate coated with 1% gelatin and cultured in M-199 me-
P M N Mediated Endothelial Damage in SLE specific release with PMN in 20% FCS medium was 1.2 f 1.5 (SD) YO.When PMN were incubated in the presence of normal sera, the mean cytotoxicity was 1.5 f 2.0 (SD) %, whereas it was 7.8 f 5.5% in the case of active SLE sera and 1.1 f 1.1% with inactive SLE. The difference between normal control and active SLE group was statistically significant (p < 0.01, Student's t-test). Four out of five sera with dermal vasculitis showed the raised cytotoxicity. SLE sera alone without PMN, as well as normal sera, were not cytotoxic during the 5-hours assay (data not shown).
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Fig. 1. Summary of the EC cytotoxicity assay mediated by PMN incubated with normal or SLE sera (active and inactive). Em ratio was 5 and incubation time was 5 hours. The horizontal line in the left column represents the value of the mean plus two standard deviations with normal sera. The difference between normal and active SLE groups was statistically significant (p < 0.01, Student's t-test). Filled circles indicate the values from sera from patients with skin vasculitis.
dium with 10% FCS for 24 hours. The medium was replaced with test serum 25 times diluted with HBSS in triplicate. After one hour at 37"C, sera were aspirated and EC were washed 3 times with HBSS. One hundred microliters of peroxidaseconjugated goat anti-human IgG, diluted at 1:4000, was added and incubated for one hour at room temperature. After washing 3 times with HBSS, 100 p1 peroxidase substrate was added to each well and incubated for 15 min. The reaction was terminated with 3 M H,S04 and the OD at 492 nm was measured by an ELISA reader. All 18 normal sera and 24 SLE sera were tested on the same day employing the same source of EC.
Gel fractionation of serum The serum samples were fractionated by gel filtration on a Sephadex G-200 column (2.7 X 100 cm) equilibrated with PBS at 4°C. One milliliter of serum was passed over the column and the fractions were collected at 5 ml/tube. Fractions for three major peaks and between the peaks were concentrated to the original volume, dialyzed against M-199 and applied to the cytotoxicity assay.
Correlation of the cytotoxicity with serum C3 levels and the circulating immune complexes Figure 2 demonstrates the correlation between the serum immune complex levels and the E C cytotoxicity-inducing activities of SLE sera. A positive significant correlation (p < 0.05) was detected between the two parameters. No significant correlation was detected between the C3 levels and activities (data not shown). Absorption of serum Two sera which showed high activity and contained a relatively large amount of immune complexes were incubated with Sepharose 4B conjugated and not conjugated with Clq. Both the original and treated sera were assessed simultaneously in the cytotoxicity assay. The dilution rates of the sera were standardized by the concentrations of albumin. As demonstrated in Fig. 3, in one serum, the E C cytotoxicity-inducing activity was decreased by 86.5% when treated with Clq-Sepharose 4B and
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Effect of sera on PMN-mediated cytotoxicity The results are demonstrated in Fig. 1. Spontaneous 51Crrelease (EC in M-199 with 20% FCS) was between 7 and 10% in the 5-hour assay and the 211
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36.5% with non-conjugated Sepharose 4B. In another serum, the results were 64.0% and 20.2%, respectively.
Correlation with anti-EC antibody Since all the sera were tested under identical conditions, values greater than mean plus 2 standard deviations of the 18 normal sera were considered as anti-EC positive. Thus, 8 out of 24 SLE sera were judged anti-EC positive and the others were negative. Figure 4 shows the relation between the presence of anti-EC antibody and the E C cytotoxicity-inducing activity. No significant difference was detected between the two groups. However, 5 out of 8 (62.5%) anti-EC posititive sera elicited an increased EC cytotoxicity-inducing activity, while 31.3% with anti-EC negative sera did. Of the five patients with dermal vasculitis, 3 were positive and the others were negative. Gel filtration of sera The three SLE sera which showed a high activity were gel filtrated on a Sephadex G-200 gel column and fractionated. Six fractions were chosen and tested for the ability to induce cytotoxicity in the assay. A normal serum was treated and applied to the assay simultaneously. In Fig. 5a-c, the results of three SLE sera are demonstrated. In the first SLE case (Fig. 5a), major activity seemed to exist in both the first and second peaks. In the second SLE case (Fig. 5b), activity mainly existed in the first peak and for the third case (Fig. 5c), in the second peak. The first and third cases were anti-EC positive and second case was negative. In contrast, no activity was detected in any fraction from the normal serum (data not shown).
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Discussion
It is well documented that polymorphonuclear leukocytes (PMN) are capable of destroying mammalian cultured cells of either tumor or non-tumor origin without accompanying phagocytosis, that is, extracellular lysis, when they are stimulated with various ligands (11, 12). It is also reported that PMN are able to destroy target cells through a specific antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism (13, 14). Endothelial cells (EC) destruction by PMN has also been demonstrated by several authors including us (9, 15, 16). The substances which stimulate PMN and initiate the cytotoxic response include chemotactic factors (15), chemical agents (16) and phagocytic stimuli including immune complexes (9). It has been suggested that this cytotoxicity may play a critical role in the immune complexmediated tissue injury, especially vasculitis. In the previous study (17), we reported that when human PMN are incubated with SLE sera they generate an elevated amount of superoxide anion and adhere more abundantly to vascular EC. We concluded that these phenomenon are ascribed to the presence of immune complexes and this we confirmed in another study (18). In the present study, we demonstrated that normal PMN, when incubated with some active SLE sera, acquired cytotoxicity against human cultured vascular EC. Inactive SLE sera as well as normal control sera did not elicit such activity. Furthermore, SLE sera in itself did not possess cytotoxic activity during the 5-hour assay. Although the degree of 51Crre-
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Fig. 5a-c. Sera from 3 SLE patients were gel fractionated over a Sephadex G-200 column. Six fractions were chosen, dialyzed, concentrated to the original volume and applied to the cytotoxicity assay. Fig. 5a,b,c represent the results of 3 patients where patients a and c were anti-EC positive and b was negative. The curved lines indicate the elution patterns monitored at OD 280 nm. Bars represent the values in the cytotoxicity assay performed using each fraction for PMN stimulation.
lease was not remarkable, we considered it to be significant as also emphasized by others (15). In consideration of possible PMN stimulatory substances in SLE sera, the correlation between this activity and either serum immune complex levels or the presence of anti-EC antibody was studied. A significant correlation was observed between the activity and the immune complex level. Furthermore, incubation of the active sera with Clq-Sepharose 4B remarkably reduced the cytotoxicity, further suggesting a contribution of circulating immune complexes in the cytotoxic activity.
The important role of immune complexes and PMN in the pathogenesis of serum sickness and so called leukocytoclastic vasculitis has been emphasized. Therefore, we speculate that this type of tissue injury mechanism may actually take place in SLE. A reduction in the activity was also observed, to a much lesser degree, in the sera treated with control Sepharose beads. We suppose that this is merely a result of the nonspecific absorption. On the other hand, no significant correlation between the activity and the presence of anti-EC was demonstrated, although anti-EC positive sera tended to induce the increased activity compared with negative sera as shown in Fig. 4.The presence of anti-EC antibody in SLE was first suggested by Shingu & Hurd (19) and was established in subsequent studies (2-5). Some of these studies showed that the presence of anti-EC antibodies correlate with vascular injury. In an animal study, furthermore, it was reported that anti-EC antibody initiates the immune complex-type glomerulonephritis (20). The results of the gel fractionation study indicated that the activity under discussion may be heterogeneous and may vary among patients. Therefore, we could not deny the possible contribution of the anti-EC antibody to cytotoxicity. In our study, it was demonstrated that the majority of the sera with dermal vasculitis contained this activity. However, it is necessary to be cadtious when discussing the relationship between the results of in vitro experiments and the clinical skin vasculitis in SLE, since it has been suggested that many other deep organ involvements, for example, CNS lupus or lupus retinopathy, are also closely related with vasculitis. In conclusion, it can be suggested that this type of E C cytotoxicity may, at least in part, contribute to the initiation of the inflammatory process of SLE and the cytotoxic activity may be mediated by circulating immune complexes and presumably by other mechanisms including anti-EC. Acknowledgement We thank Miss Kyoko Shimada for her excellent technical assistance. We also wish to thank the staff of the Tokyo Metropolitan Tsukiji Obstetric Hospital for providing umbilical cords. Supported by Manabe Medical Foundation.
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