Journal of Clinical Immunology, Vol. 22, No. 4, July 2002 (©2002)
Serum sFAS Levels Are Elevated in ANCA-Positive Vasculitis Compared with Other Autoimmune Diseases ¨ TT,3 BIRGER CHRISTENSSON,1,4 MARTA CHRISTENSSON,1,7 ERNA PETTERSSON,2 KJELL ENESLA 5 6 ¨A ¨ -DAHLQVIST, and KARL-GO ¨ STA SUNDQVIST3 JOHAN BRATT, SOLBRITT RANTAPA
the vasculitis patients showed an increased mRNA expression of Fas in mononuclear cells after treatment, suggesting that Fas production fluctuates with the intensity of the disease. The expression of CD95 on leukocytes was slightly decreased in vasculitis patients compared to healthy controls. No alterations of Fas and FasL expression were seen in renal biopsy specimens. These results show that ANCA-positive vasculitis patients have high sFas levels and that the levels remain elevated even in clinical remission. The findings indicate that perturbations in the Fas/Fas ligand system may play a role in the disease process in ANCA vasculitis.
Accepted: February 6, 2002
The role of the Fas/FasL system in ANCA-associated vasculitis is unclear. We therefore assessed levels of soluble Fas (sFas) in sera and Fas expression on mononuclear cells from patients with ANCA-positive vasculitis and compared the results with those found in other rheumatic diseases. Serum levels of sFas were determined by ELISA. The ANCA-positive vasculitis patients studied included 29 at onset, 17 in first remission while on therapy, and 12 in quiescence. For comparison, 10 patients with Sjo¨gren’s syndrome (SS), 14 patients with systemic lupus erythematosus (SLE), 29 patients with rheumatoid arthritis (RA), 7 patients on dialysis (DP), and 26 healthy controls (HC) were studied. In addition, Fas expression in mononuclear cells was examined at the mRNA level using reverse transcriptase (RT)-PCR in 6 vasculitis patients at onset and in first remission. The expression of CD95 on the surface of leukocytes was determined by flow cytometry in 6 vasculitis patients at onset of the disease, in 6 patients in clinical remission, and in 6 HC. Expression of Fas and FasL in renal biopsy specimens was studied using immunohistochemistry. Patients with vasculitis had high sFas levels irrespective of disease phase. Both vasculitis patients and patients with RA and SLE had significantly increased sFas levels compared with healthy controls. All patient groups had sFas levels, which correlated with raised serum creatinine values. However, the sFas levels in vasculitis patients in first remission and in quiescence were increased despite a lower serum creatinine compared with onset. Some of
KEY WORDS: Fas; vasculitis; therapy.
INTRODUCTION
There is increasing evidence that defects in apoptosis may be responsible for the development of autoimmunity (1, 2). The involvement of the Fas/FasL system in the regulation of apoptosis was shown in the lpr and lprcg mouse strains where mutations in the lpr and gld genes led to deficiency in the expression of Fas and Fas ligand (3, 4). As a consequence, the elimination of autoreactive cells and the down-regulation of lymphocyte responses are defective. In the human disease autoimmune lymphoproliferative syndrome (ALPS), mutational changes in the Fas and FasL molecules lead to specific forms of autoimmunity, to an accumulation of lymphoid cells, and to a higher incidence of lymphoid malignancy (2, 5). Fas is produced by many cell types and released from cells by diverse mechanisms and may therefore be detected as a soluble form (sFas) in serum. Elevated sFas levels in serum may reflect release from vascular endothelial cells or from activated leukocytes and is thought to be a mechanism to protect various cells from FasL-mediated damage (6). The notion that perturbations in the Fas/FasL system may be more generally responsible for the development of autoimmune diseases has prompted studies in sys-
1
Department of Clinical Immunology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. Department of Renal Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. 3 Department of Clinical Immunology, Umeå University, Umeå, Sweden. 4 Department of Pathology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. 5 Department of Rheumatology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. 6 Department of Rheumatology, Umeå University, Umeå, Sweden. 7 To whom correspondence should be addressed at Department of Clinical Immunology F79, Huddinge University Hospital, S 141 86 Stockholm, Sweden. Fax: 46-8-5858 1390; email: marta.christensson@ immunlab.hs.sll.se 2
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temic lupus erythematosus (SLE) (2, 7, 8). The results so far have been controversial. Jodo et al. (9) propose that sFas levels may be used as a marker of disease activity, and Kovacs et al. (10) find increased expression of functional FasL in T cells from SLE patients. Furthermore, McNally et al. (11) conclude that no specific defect in FasL function are found in SLE patients, and Knipping et al. (12) propose that the role of sFas in the pathogenesis of SLE is questionable. Impairment of renal function seems to be associated with increased sFas levels, and Sato et al. (13) suggest that the expression of Fas antigen on mononuclear cells in peripheral blood could be activated as a consequence of deterioration in renal function. In the spectrum of ANCA-associated vasculitides, a generalized inflammatory activity is accompanied by vascular lesions in various organs. The pathogenesis is largely unknown in spite of distinct autoantibody patterns. Both nonspecific and T-cell-mediated immune mechanisms have been invoked in the development and perpetuation of the disease (14, 15). We and others have previously found that patients with ANCA-positive vasculitis show an increased expression of T-cell activation markers irrespective of immunosuppressive therapy or disease phase (16, 17). The possible role of apoptotic mechanisms in ANCA-associated vasculitis in unclear. To investigate whether the Fas/FasL system is involved in the autoimmune vascular disease we measured both serum levels of sFas and FasL in patients with ANCApositive vasculitis at different stages of the disease and the expression of Fas/FasL on peripheral mononuclear cells and in kidney biopsy specimens. The results were compared with those of other autoimmune diseases and healthy controls.
MATERIALS AND METHODS
Patients Thirty-seven patients with ANCA-positive vasculitis from the Departments of Nephrology and Rheumatology at Huddinge University Hospital were included. The ANCA-positive vasculitides were defined according to the Chapel Hill Consensus on the Nomenclature of Systemic Vasculitides for the following disease entities: Wegener’s granulomatosis (WG), clinical Wegener’s (cWG), and microscopic polyangiitis (MPA) (18). In addition, ANCA-positive vasculitides included patients having pauci-immune necrotizing glomerulonephritis with or without crescents (CNGN) referred to as renal limited disease (RLD) and patients with only upper
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airway involvement, addressed as limited form of Wegener’s granulomatosis (IWG). Disease activity was evaluated with the Birmingham vasculitis activity score (BVAS) (19). The patients were categorized according to the phase of their disease (see Table I). Twenty-nine (ages 28 – 83 years) had a newly diagnosed active disease. Blood samples were collected before initiation of immunosuppressive therapy. Seventeen of these patients were reanalyzed 3–11 months later while in first clinical remission. Twelve other patients (ages 48 –77 years) were studied in quiescence (2–10 years after disease onset). Nine of them showed neither clinical nor inflammatory (CRP ⬍ 10) activity, while two had persistent, low-grade “grumbling” disease and slight inflammatory activity (CRP ⬍ 50) with symptoms from the upper airways but no evidence of infection. At the time of analysis, four patients were on long-term methotrexate and two were on cyclophosphamide remedy; six received no immunosuppression at all. For comparison, sFas in serum was analyzed in patients with other autoimmune diseases. Twenty-nine patients with rheumatoid arthritis (RA), (ages 24 – 83 years) and median disease duration 11.0 years (range, 0.5–37 years) had a mean accumulated disease activity score of 5.7⫹/⫺0.3 (20, 21). Eleven of them were on corticosteroids and all except two were on diseasemodifying antirheumatic drugs, e.g., sulphasalazine, oral or parenteral gold, antimalarials, methotrexate, azathioprine, or penicillamine. Of 14 patients with SLE (ages 18 –72 years) with median disease duration of 5.0 years (range, 0.5–32 years), 1 had high disease activity, 4 had medium disease activity, and 7 had low disease activity (22–24). Two were receiving pulse doses of cyclophosphamide and three were on azathioprine, three on antimalarials, one on methotrexate, and another on cyclosporine. Eleven of them were on corticosteroids. Ten patients had Sjo¨ gren’s syndrome (SS) (ages 35–73 years) with a median disease duration of 11.5 years (range, 2–20 years) (25). All the patients had low disease inflammatory activity; one of them was on antimalarials. Seven stable patients on dialysis (ages 32–78 years) were included in order to study the influence of advanced renal impairment on sFas levels. None of them fulfilled the criteria for vasculitis, SLE, or other autoimmune disease. Twenty-six healthy controls were included (ages 26 –78 years). EDTA venous blood, heparin blood, and serum samples were collected at the time of diagnosis and at routine visits to the outpatient clinics.
a
96 151 106 76 67 113 77 161 99 188 190 103
No No CP CS⫹MTX CS⫹MTX CS⫹CP MTX No No No No MTX
11400 7835 12900 11700 8197 17900 15900 10100 4033 7550 17900 nd 15400 12200 17500 6487 14100 26400 15100 9011 12800 18500 2576 13300 15500 13950 15150 17200 13700 13100 13400 24700 12400 10000 15100 9606 12900 10390 17800 25700 14900
⬍10 ⬍10 nd (ERS ⫽ 57) nd (ERS ⫽ 17) nd (ERS ⫽ 8) 49 nd (ERS ⫽ 3) ⬍10 14 ⬍10 ⬍10 ⬍10 No No Low no no low no no no no no no
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
95
60 210 80 nd 250 ⬍10 15 ⬍10
37
300 50 40
100
120 ⬍10 100 45 170 120 35
290 70
35 65
45 ⬍10 300
nd
300 125 80 80 90 500 75
Group 2: At follow up CS⫹CP 82 CS⫹CP 81 80 CS⫹CP 224 CS⫹CP 121 CS⫹MTX 80 20 CS⫹CP 197 CS 892 ⬍10 CS⫹Aza 110 CS⫹CP 62 CS⫹CP 141 CS⫹CP 295 CS⫹CP 88 CS⫹CP 188 40 no 105 245 CS⫹Aza 257 CS⫹CP 100 390 CS 1070 35 CS⫹CP 186 CS⫹Aza 138
Disease PR3 MPO Creatinine sFas(pg/ml) activity units units Treatment mole/liter
21 61 104 ⬍10 14 131 148 151 203 70 150 136 ⬍10 ⬍10 21 145 10 57 ⬍10 107 38 29 207 37 92 186 53 32 107
CRP ⬍10 17 73 ⬍10 ⬍10 21 ⬍10 ⬍10 nd (ESR⫽9) ⬍10 ⬍10 nd ⬍10 ⬍10 ⬍10 ⬍10 222 ⬍10 ⬍10
CRP nd 7938 12500 10900 7638 nd 18700 10400 9355 18200 20900 14200 17200 19200 19500 10000 21700 18100 16100
Low Low Low no no no no* no no no no no no no no no no* no No
⬍10
⬍10
25 ⬍10 ⬍10 15 15 20 ⬍10
85 20
20 25
80 ⬍10
25 ⬍10
⬍10
40
⬍10
Disease PR3 MPO sFas(pg/ml) activity units units
*, Dialysis; CS, corticosteroids; CP, cyclophosphamide; MTX, methotrexate; Aza, azathioprine; nd, nondetermined; MPA; microscopic polyangiitis; RLD, renal limited disease; cWg, clinical Wegener’s; Wg, Wegener’s; lWg, limited Wegener’s; ESR, erythrosedimentation rate; F, female; M, male.
73 69 458 131 83 136 486 812 51 265 594 93 176 103 534 82 478 411 175 73 220 336 289 234 83 681 383 255 319
No No No CS No No CS No No No No No No No No No No No CS CS CS No No CS CS No No No No
Creatinine Diagnosis Treatment mole/liter
Group 1: At admission 1. 48, F Wg 2. 70, F cWg 3. 65, F MPA 4. 69, M cWg 5. 24, F lWg 6. 76, M Wg 7. 54, M cWg 8. 45, M Wg 9. 46, F Wg 10. 69, M cWg 11. 49, M RLD 12. 77, F cWg 13. 67, M cWg 14. 70, F lWg 15. 63, F RLD 16. 28, M Wg 17. 40, M MPA 18. 75, M Wg 19. 37, M MPA 20. 73, M Wg 21. 57, F Wg 22. 76, M MPA 23. 81, F MPA 24. 77, F MPA 25. 77, F Wg 26. 83, F RLD 27. 70, F RLD 28. 81, M MPA 29. 60, M MPA Group 3: In quiescence 30. 73, M Wg 31. 60, M cWg 32. 75, M cWg 33. 49, M Wg 34. 48, F Wg 35. 59, M cWg 36. 53, M Wg 37. 56, F Wg 38. 67, F cWg 39. 61, F cWg 40. 49, M MPA 41. 77, M Wg
Patient age/sex
Table I. Some Clinical and Laboratory Data of the Vasculitis Patientsa
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sFas and sFasL Analysis Serum levels of sFas and FasL were assessed by ELISA, using kits from R&D Systems (Minneapolis, MN) according to the protocol recommended by the producer. Analysis of Lymphocyte Subsets and CD95 Expression In order to study the surface expression of CD95 (Fas) on peripheral blood cells, the frequency of CD95 positive cells as well as the intensity of the CD95 expression was analyzed by flow cytometry in lymphocytes, monocytes, and neutrophils in six patients with ANCA-positive vasculitis at disease onset. These patients also were analyzed after initial treatment. As well, seven patients on dialysis without inflammatory/autoimmune disease and six healthy controls were included. Three-color immunofluorescence analysis was performed according to standard procedures for flow cytometry of whole blood samples, using a “lyse and wash” protocol. In short, 100 l of EDTA blood was mixed with appropriate concentrations of directly fluorochrome-conjugated monoclonal antibodies; the lymphocyte/monocyte markers CD45/14 and antigens on T cells (CD3, CD4, CD8) and antibodies to Fas/CD95 (APO1) were used. All antibodies were obtained from BectonDickinson (Mountain View, CA). After addition of the primary antibody the blood samples were incubated for 15 min at room temperature and then washed in phosphate-buffered saline pending analysis. Data were acquired on a FACScan (Becton-Dickinson), using the Cellquest software both for acquisition and analysis. For each sample 3000 lymphocytes were acquired, using log-amplified fluorescence and linearly amplified sideand forward-scatter signals. All samples were analyzed by setting appropriate SSC/FSC gates around the lymphocyte population, using back gating on CD45 positive, CD14-negative cells. Consistency of analysis parameters was ascertained by calibrating the flow cytometer with Calibrite beads and the AutoComp software, both from Becton-Dickinson.
Molecular Systems, Inc., NJ). RT-PCR was performed at 42°C for 15 min followed by denaturation at 99°C for 5 min. The following primers were used: (a) Fas receptor: 5⬘-TCTTTCACTTCGGAGGATTGCT-3⬘; 5⬘-ACTTTCTGTTCTGCTGTGTCTT-3⬘ [3]; (b) -actin: 5⬘-GTGGGGCGCCCAGGCACCA-3⬘; 5⬘-CTCCTTAATGTCACGCACGATTTC-3⬘. Twenty microliters of the cDNA reaction mixture and the specific primers were used with standard protocol to amplify the 832 bp (Fas) and 540 bp -actin fragments. PCR products were analyzed on 1.5% agarose gels, stained with ethidium bromide and analyzed under UV light. Immunohistochemistry Paraffin sections of kidney biopsies from five cases each of vasculitis, SLE, and IgA nephropathies, respectively, were analyzed immunohistochemically for Fas and FasL expression. The following monoclonal antibodies (MAbs) were used: Fas [CD95, clones APO-1 and DX2 (Dakopatts, Glostrup, Denmark)] and Fas Ligand [CD95L, clone 5D1 (NovoCastra Lab, Newcastle, UK)]. The endogenous peroxidase activity was blocked. The antigen retrieval processes were used for Fas and CD95L staining. The immunohistochemical staining, a streptavidin– biotin procedure, was performed in a DAKO TechMate 500 PLUS, using DAKO ChemMate solutions (Dakopatts). Statistical Analysis Correlation was calculated by Spearman rank-order correlation (rs). The Wilcoxon rank sum-test was used to compare data between groups (the Kruskall–Wallis test was used first). P values of ⬍ 0.05 were considered significant.
RESULTS
RNA Extraction and RT-PCR for Fas
Serum Levels of Soluble Fas (sFas) and of Soluble Fas Ligand
Total RNA was isolated from PBMC using the guanidinium thiocyanate-phenol chloroform method (26). The RNA was treated with RNase inhibitor (Rnasin, Promega, Madison, WI), 1 l/50 l RNA and stored at ⫺70°C if not used immediately. Single-stranded cDNA copies were made from 1 g of total RNA using random hexamers and murine leukemia virus transcriptase (Perkin-Elmer, Roche
The results of sFas analysis are summarized in Tables I and II and Fig. 1a. The vasculitis patients and patients with RA and SLE had significantly increased sFas levels compared with healthy controls. The median serum levels of sFas in the vasculitis patients were high irrespective of disease phase in comparison with other patient groups (Table II). Paired analysis of sFas levels at
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Table II. sFas and Creatinine Levels in the Patient Groups and Healthy Controls s-creatinine (mol/liter)
sFas (pg/ml) a
Disease
median
(25%–75%)
median
(25%–75%)
Vasculitis Onset Remission Quiescence RA SS SLE HC
13700 15150 13250 8650 7770 8800 7400
9000 –15900 10100 –18600 10900 –17100 6900 –11900 6150 –9500 7300 –12350 6500 –9000
255 130 105 80 78 70 100
80 – 460 90 –220 80 –160 70 – 85 75–95 60 –90 80 –110
a
RA, Rheumatoid arthritis; SS, Sjo¨ grens syndrome; SLE, lupus systemic erythematosus; HC, healthy controls.
onset and first remission in 15 vasculitis patients and serum creatinine values are shown in Fig. 1a, b. All patient groups had sFas levels which correlated with raised serum creatinine values (r ⫽ 0.56, P ⬍ 0.05). In fact, stable dialysis patients had the highest levels (median: 20,000 pg/ml, 25/75%: 18,300/22,700). However, in vasculitis patients the sFas levels in first remission and in quiescence were increased despite lower serum creatinine levels (Fig. 1a, b and Tables I and II) compared with patients at onset.
Soluble FasL levels in vasculitis patients at disease onset were similar to those of healthy controls (0.09 ng/ml). Patients with vasculitis in quiescence had slightly higher levels of sFasL (0.10 ng/ml) than patients at onset and in remission, as well as the healthy controls, but the difference was not significantly (data not shown). Surface Expression of CD95/Fas-r The level of expression of CD95 on the surface of monocytes and neutrophils was slightly lower in six vasculitis patients at onset [mean fluorescence intensity (MFI): median 79, 135, 102, respectively, and after first remission (MFI: median 89, 146 and 124 respectively)] compared with healthy controls (MFI: 75, 172, 133, respectively), while the expression of CD95 on lymphocytes was slightly higher (Table III). Patients on dialysis showed lower expression of CD95 on granulocytes, lymphocytes, and monocytes than healthy controls (Table III). When compared to the vasculitis group after first remission, patients on dialysis showed lower CD95 expression on granulocytes and lymphocytes, while CD95 expression on monocytes was equal for both groups. However, the differences were not statistically significant (data not shown). Fas mRNA Expression
Fig. 1. sFas (a) and s-creatinine (b) levels in 15 patients with ANCA-positive vasculitis at onset and in first remission (mean ⫹/⫺ SE). sFas was assessed by ELISA and s-creatinine by Jasse´ ’s method.
The expression of Fas in mononuclear cells was also examined at the mRNA-level by RT-PCR from six patients at onset (11, 12, 14, 15, 17, 19 in Table I) and after treatment (Fig. 2). All vasculitis patients expressed Fas mRNA. Patient 14 showed a weak expression of Fas at onset, while at follow up the expression was markedly higher. In addition to the higher Fas expression, three splicing forms of Fas (770 kb, 724 kb, and 586 kb) designated b, c, and d, respectively also appeared in patient 14 at follow up (lane 6). The splicing forms were not visible in the sample taken at onset of disease from
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Table III. MFI for CD95 and sFas in Patients with Vasculitis, in Dialysis Patients, and in Healthy Controls Disease
MFI lymphocytes median (25–75%)a
MFI monocytes median (25–75%)
MFI granulocytes median (25–75%)
sFas pg/ml median (25–75%)
Vasculitis at onset Dialysis patients Healthy controls
79 (70 –97) 69 (41– 83) 75 (52–92)
135 (115–162) 147 (122–189) 172 (140 –222)
102 (78 –123) 102 (69 –145) 133 (106 –160)
11550 (8197–17300) 20000 (15000 –22700) 7366 (6500 –9000)
a
MFI, mean fluorescence intensity.
the same patient. Patients 11 and 15 also had a weak expression of Fas at onset but a strong expression after treatment. However, no splice forms were seen in 11 and 15. It is most likely that the appearance of the splicing forms after treatment is due to an increased expression of Fas. Immunohistochemistry The expression of Fas and FasL in renal biopsy specimens were similar irrespective of the pathoanatomical diagnosis (vasculitis, SLE, or IgA nephropathy). Some segments of the tubular system were consistently Fas positive. In Bowman’s capsule, a variable, rather weak Fas staining could occasionally be seen. In the glomeruli, only some mesangial cells were stained for FasL. In some cases arterial endothelial cells were weakly positive. Some infiltrating leukocytes and plasma cells were also FasL positive (data not shown).
DISCUSSION
Clonal down-regulation of immune responses appears to be one of the critical ways of abrogating an immune reaction. In vitro studies have indicated that this process may be mediated by T cells expressing CD95 and CD95L; thus, the trimerization of the Fas/CD95 receptor by its ligand mediates apoptosis of sensitive cells. The presence of an excess of sFas could potentially interfere with this process, and thus lead to an accumulation of antigen-specific T cells similar to that seen in the lpr and gld mouse models of autoimmunity and in autoimmune lymphoproliferative syndrome first suggested for SLE by Cheng et al. (27). The soluble form of sFas has been suggested to offer protection from Fas-mediated apoptosis (27). This concept is supported by the finding that patients with SLE, polymyositis/dermatomyositis, mixed connective tissue disease, and SS show elevated levels of sFas (28). Nozawa et al. (28) were able to demonstrate a relation-
Fig. 2. RT-PCR analysis of human Fas mRNA transcripts obtained from unstimulated PBL from 6 vasculitis patients before treatment (lanes 1, 3, 5, 7, 9, 11) and after treatment (lanes 2, 4, 6, 8, 10, 12) (patient 19, lanes 1 and 2; patient 11, lanes 3 and 4; patient 14, lanes 5 and 6; patient 15, lanes 7 and 8; patient 12, lanes 9 and 10 and patient 17, lanes 11 and 12). First-strand cDNA prepared from PBL were subjected to PCR with the primer pair described in material and methods. The PCR product was separated on a 1.5% agarose gel and visualized by ethidium bromide staining and UV illumination. All patients expressed Fas mRNA. In addition to the full-length Fas transcript (a), three splicing forms of Fas (b, c, and d) also appeared in patient 14 at follow up (lane 6). The upper band in the different lanes is an extra band that does not represent any known Fas transcript. Patients 11 and 15 also had a weak expression at onset but a stronger Fas expression after treatment. “St” indicates the molecular weight markers; “⫺” represents negative control.
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ship between disease activity in SLE patients and sFas levels, while two other groups (12, 29) did not find a relationship between sFas levels and disease activity in SLE and RA. Al-Maini et al. (6) found a correlation between sFas and organ damage in SLE but not in the case of disease activity. The present results confirm and extend results from earlier studies demonstrating that patients with rheumatic diseases show elevated levels of sFas (28). Our study also confirms the finding by Sato et al. (13) that sFas levels increase with deteriorating renal function. Therefore, any analysis of sFas levels in patients should be related to renal function. The highest sFas levels were seen in our vasculitis patients, and interestingly the sFas levels tended to rise as disease activity decreased and renal function improved. Possibly, the mechanisms generating sFas elevation in vasculitis are more difficult to suppress than in other autoimmune diseases (30). To elucidate whether the elevated sFas levels might originate from leukocytes, we examined CD95 in lymphocytes, granulocytes, and monocytes of patients with vasculitis at onset and compared its expression with that of normal controls and patients on dialysis without known inflammatory disease. All cell types from vasculitis patients showed lower CD95 expression than the corresponding cells from healthy controls (Table III). In contrast to the findings of Sato et al. (13), we found that patients on dialysis showed even lower expression of CD95 on leukocytes compared to the vasculitis patients. The apparent inverse relationship between serum sFas level and cell surface expression of Fas may have several explanations: (a) Fas serves as a competitive inhibitor of the CD95 antibody, and thus gives a decreased staining intensity. However, by adding patient serum with high sFas levels to control leukocytes, no significant decrease in CD95 staining was seen (unpublished data). (b) Increased sFas levels are associated with an increased turnover of CD95 giving lower staining intensity. (c) Increased sFas is associated with a switch from membrane bound to soluble Fas. The fact that three patients showed an increased Fas mRNA expression after treatment may reflect treatment effects on Fas expression per se or appearance of lymphocyte populations with different Fas expression in the blood. However, no correlation between sFas levels and leukocyte count was found. The cellular origin of serum sFas is not well understood. The possibility that sFas is generated in the tissues
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most affected by the vasculitis disease was addressed immunohistochemically. Nevertheless, no apparent increase in Fas protein expression in renal biopsies as detected by two CD95-specific antibodies could be visualized. In contrast, leukocytes from vasculitis patients showed a reduced surface expression of Fas, which points to the possibility that sFas in serum may be derived from leukocytes. In conclusion, this study showed an increase in sFas levels in vasculitis patients even in clinical remission. This may be an indicator of a persisting chronic immune activation in concordance with our earlier studies on T cells (16). Suppression of this underlying immune activation may be an important target for novel therapies in relapse prevention.
ACKNOWLEDGMENTS
We thank Dr. Mikael Heimbu¨ rger for excellent clinical collaboration and for providing patients’ samples and Dr. Magnus So¨ derberg for his help with the biopsies. This research study was supported by the Swedish Medical Research Council (Project 16x-08295) and the Karolinska Institute, Stockholm, Sweden.
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