Autoimmunity, 1992, Vol. 1 I . pp. 213-214 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
Correspondence
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/28/14 For personal use only.
CIRCULATING AUTOANTIBODIES DIRECTED AGAINST P-GLUCURONIDASE Sir, Autoantibodies directed against granulocyte lysosoma1 enzymes have recently been described in patients presenting with systemic vaculitis. Antineutrophil cytoplasm antibodies (c-ANCA) were found in Wegener's granulomatosis' and in related disorders with systemic vasculitis*. Antibodies against myeloperoxidase (anti-MPO) and elastase (antielastase) have also been detected in patients with vasculitis especially those involving the kidney. We have previously reported an anti-MPO frequency of 25% in patients with crescentic gl~merulonephritis~. Furthermore, we have found anti-elastase as well as anti-MPO to be markers in hydralazine-induced SLE syndrome4. The contribution of these autoantibodies to the underlying pathogenetic mechanisms is unclear. Whether these new autoantibodies play a role for the development of the disease or not, it might be realistic to assume the possibility of circulating antibodies against other lysosomal enzymes. It has been reported that p-glucuronidase, a lysosomal enzyme also found in human neutrophil granulocytes and harboured in the same granula as myeloperoxidase, is secreted in parallel with myeloperoxidase during stimulation with f-Met-Leu-Phe and cytochalasin B5. Recently, it has been shown that complement together with immune complex induce release of p-glucuronidase by polymorphonuclear leucoytes'. Furthermore, on immunofluorescence we have found patterns which do not correspond to autoantibodies known today. We have developed an ELISA for detection of antibodies to P-glucuronidase. Briefly, 1 pug p-glucuronidase (G0501, Sigma Chemical) was coated per well (Nunc Maxisorb, Roskilde, DK). Sera were applied for 2 hr in dilution 1/100 in PBS-O.OS% Tween 20. After washing, F(ab')2 rabbit antihuman IgG alkaline phosphatase conjugated (1/750) was added for 2 hr thereafter substrate incubation during 45 min followed by spectrophotometric reading at 405 nm. We investigated 29 patients with anti-MPO, 4 patients with c-ANCA, and 13 patients with a peripheral or segmentary unclear immunofluorescence pattern negative for anti-MPO and anti-elastase. Fifty sera sampled from normal blood donors were included in this study.
The cut off level in the assay was set at 0.20 (0.1l8k2.5 SD). Circulating autoantibodies to pglucuronidase of IgG-class were found in 5 patients in the anti-MPO group (Figure l), but no correlation to the anti-MPO titre was seen. Seven sera in the normal blood donor group had absorbance values above 0.220 and therefore they were excluded as controls. However, the highest titres of anti-p-glucuronidase were found in the anti-MPO group (Figure 1).
/q 0.8-
E
c I n 0
d
Q)
0.6-
0
c m
nL
-
0
ln
n
4 0.4-
213
0 0
: O.*I
- -
NS
+anti-MPO
-anti-MPO
Figure 1 Distribution of autoantibodies directed against pglucuronidase in relation to positive and negative anti-MPO sera, respectively. The dotted line indicates cut off level for controls (mean+2.S SD). NS: Sera from normal blood donors. +anti-MPO: scra containing circulating anti-MPO. -anti-MPO: sera without circulating anti-MPO.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/28/14 For personal use only.
214
CORRESPONDENCE
It was possible to neutralize all positive sera in the fluid phase by preincubation with a surplus of 50 p1 P-glucuronidase (0.1 mg/ml) with 50 pl serum diluted 1/50 for 60 min and subsequent incubation in the ELISA performed as described above. Very interesting was the finding of these autoantibodies in healthy sera. Antibodies in normal healthy sera have been reported to occur in a high freq~ency’.~ but the significance is unclear. Neutrophil granulocytes are participating in the defence against invading microrganisms and release of lysosomal enzymes may take place. They may undergo antigen processing in antigen presenting cells giving rise to antibody production. The importance of these antibodies against Pglucuronidase from a clinical point of view remains to be elucidated, and it is at the moment impossible to subscribe these autoantibodies to a special subtype of vasculitis. However, it also seems to be the case that circulating autoantibodies against lysosoma1 enzymes do not necessarily reflect an ongoing disease process, since we found circulating autoantibodies also in healthy individuals. U. MARTENSSON and L. NASSBERGER Department of Medical Microbiology, Clinical Immunology, University of Lund, S-223 62 Lund, Sweden Correspondence: Lennart Nlssberger, Department of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
References 1. van der Woude FJ, Lobatto S, Permin H, van der Giessen J, Rasmussen N, Wiik A, et al. Autoantibodies against neutro-
2.
3. 4.
5. 6.
7.
8.
phils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet 1985; i: 524-529 Nlssberger L, Sjoholm AG, Bygren P, Thysell H, Madsen MH, Rasmussen N. Circulating antineutrophil cytoplsm antibodies in patients with rapidly progressive glomerulonephritis and extracapillary proliferation. J Intern Med 1989; 225: 791-796 Nlssberger L, Sjoholm AG, Thysell H. Antimyeloperoxidase antibodies in patients with extracapillary glomerulonephritis. Nephron 1990; 56: 152-156 Nassberger L, Johansson AC, Bjorck S, Sjoholm AG. Antibodies to neutrophil granulocyte myeloperoxidase and elastase: autoimmune responses in glomerulonephritis due to hydralazine treatment. J Intern Med 1991; 229: 261-265 Edwards SW, Nurcombe HL, Hart CA. Oxidative inactivation of myeloperoxidase released from human neutrophils. Biochem J 1987; 245: 925-928 Lucisano A, Mantovani B. The role of complement in the stimulation of lysosomal enzyme release by polymorphonuclear leucoytes induced by immune complexes of IgG and of IgM. Immunology 1988; 65: 171-175 Shapiro PE, McAllister G, Lemer EA. Healthy individuals and patients with systemic lupus erythematosus have unique, specific spectra of antibodies detectable on immunoblots. Clin lmmun Immunopath 1991; 59: 129-138 Osung OA, Chandra M, Holborow EJ. Antibody to intermediate filaments of the cytoskeleton in rheumatoid arthritis. Annals of the Rheumatic Diseases 1989; 41: 69-73
0 1992 Harwood Academic Publishers GmbH Printed in the United Kingdom
Correspondence
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/28/14 For personal use only.
CIRCULATING AUTOANTIBODIES DIRECTED AGAINST P-GLUCURONIDASE Sir, Autoantibodies directed against granulocyte lysosoma1 enzymes have recently been described in patients presenting with systemic vaculitis. Antineutrophil cytoplasm antibodies (c-ANCA) were found in Wegener's granulomatosis' and in related disorders with systemic vasculitis*. Antibodies against myeloperoxidase (anti-MPO) and elastase (antielastase) have also been detected in patients with vasculitis especially those involving the kidney. We have previously reported an anti-MPO frequency of 25% in patients with crescentic gl~merulonephritis~. Furthermore, we have found anti-elastase as well as anti-MPO to be markers in hydralazine-induced SLE syndrome4. The contribution of these autoantibodies to the underlying pathogenetic mechanisms is unclear. Whether these new autoantibodies play a role for the development of the disease or not, it might be realistic to assume the possibility of circulating antibodies against other lysosomal enzymes. It has been reported that p-glucuronidase, a lysosomal enzyme also found in human neutrophil granulocytes and harboured in the same granula as myeloperoxidase, is secreted in parallel with myeloperoxidase during stimulation with f-Met-Leu-Phe and cytochalasin B5. Recently, it has been shown that complement together with immune complex induce release of p-glucuronidase by polymorphonuclear leucoytes'. Furthermore, on immunofluorescence we have found patterns which do not correspond to autoantibodies known today. We have developed an ELISA for detection of antibodies to P-glucuronidase. Briefly, 1 pug p-glucuronidase (G0501, Sigma Chemical) was coated per well (Nunc Maxisorb, Roskilde, DK). Sera were applied for 2 hr in dilution 1/100 in PBS-O.OS% Tween 20. After washing, F(ab')2 rabbit antihuman IgG alkaline phosphatase conjugated (1/750) was added for 2 hr thereafter substrate incubation during 45 min followed by spectrophotometric reading at 405 nm. We investigated 29 patients with anti-MPO, 4 patients with c-ANCA, and 13 patients with a peripheral or segmentary unclear immunofluorescence pattern negative for anti-MPO and anti-elastase. Fifty sera sampled from normal blood donors were included in this study.
The cut off level in the assay was set at 0.20 (0.1l8k2.5 SD). Circulating autoantibodies to pglucuronidase of IgG-class were found in 5 patients in the anti-MPO group (Figure l), but no correlation to the anti-MPO titre was seen. Seven sera in the normal blood donor group had absorbance values above 0.220 and therefore they were excluded as controls. However, the highest titres of anti-p-glucuronidase were found in the anti-MPO group (Figure 1).
/q 0.8-
E
c I n 0
d
Q)
0.6-
0
c m
nL
-
0
ln
n
4 0.4-
213
0 0
: O.*I
- -
NS
+anti-MPO
-anti-MPO
Figure 1 Distribution of autoantibodies directed against pglucuronidase in relation to positive and negative anti-MPO sera, respectively. The dotted line indicates cut off level for controls (mean+2.S SD). NS: Sera from normal blood donors. +anti-MPO: scra containing circulating anti-MPO. -anti-MPO: sera without circulating anti-MPO.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/28/14 For personal use only.
214
CORRESPONDENCE
It was possible to neutralize all positive sera in the fluid phase by preincubation with a surplus of 50 p1 P-glucuronidase (0.1 mg/ml) with 50 pl serum diluted 1/50 for 60 min and subsequent incubation in the ELISA performed as described above. Very interesting was the finding of these autoantibodies in healthy sera. Antibodies in normal healthy sera have been reported to occur in a high freq~ency’.~ but the significance is unclear. Neutrophil granulocytes are participating in the defence against invading microrganisms and release of lysosomal enzymes may take place. They may undergo antigen processing in antigen presenting cells giving rise to antibody production. The importance of these antibodies against Pglucuronidase from a clinical point of view remains to be elucidated, and it is at the moment impossible to subscribe these autoantibodies to a special subtype of vasculitis. However, it also seems to be the case that circulating autoantibodies against lysosoma1 enzymes do not necessarily reflect an ongoing disease process, since we found circulating autoantibodies also in healthy individuals. U. MARTENSSON and L. NASSBERGER Department of Medical Microbiology, Clinical Immunology, University of Lund, S-223 62 Lund, Sweden Correspondence: Lennart Nlssberger, Department of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
References 1. van der Woude FJ, Lobatto S, Permin H, van der Giessen J, Rasmussen N, Wiik A, et al. Autoantibodies against neutro-
2.
3. 4.
5. 6.
7.
8.
phils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet 1985; i: 524-529 Nlssberger L, Sjoholm AG, Bygren P, Thysell H, Madsen MH, Rasmussen N. Circulating antineutrophil cytoplsm antibodies in patients with rapidly progressive glomerulonephritis and extracapillary proliferation. J Intern Med 1989; 225: 791-796 Nlssberger L, Sjoholm AG, Thysell H. Antimyeloperoxidase antibodies in patients with extracapillary glomerulonephritis. Nephron 1990; 56: 152-156 Nassberger L, Johansson AC, Bjorck S, Sjoholm AG. Antibodies to neutrophil granulocyte myeloperoxidase and elastase: autoimmune responses in glomerulonephritis due to hydralazine treatment. J Intern Med 1991; 229: 261-265 Edwards SW, Nurcombe HL, Hart CA. Oxidative inactivation of myeloperoxidase released from human neutrophils. Biochem J 1987; 245: 925-928 Lucisano A, Mantovani B. The role of complement in the stimulation of lysosomal enzyme release by polymorphonuclear leucoytes induced by immune complexes of IgG and of IgM. Immunology 1988; 65: 171-175 Shapiro PE, McAllister G, Lemer EA. Healthy individuals and patients with systemic lupus erythematosus have unique, specific spectra of antibodies detectable on immunoblots. Clin lmmun Immunopath 1991; 59: 129-138 Osung OA, Chandra M, Holborow EJ. Antibody to intermediate filaments of the cytoskeleton in rheumatoid arthritis. Annals of the Rheumatic Diseases 1989; 41: 69-73
