IgG Subclasses in
Systemic
Lupus Erythematosus and Other
Autoimmune Rheumatic Diseases F.
BLANCO*,
J.
KALSI*,
C.T.
RAVIRAJAN*,
P.
SPEIGHT**,
A.R. BRADWELL † and D.A. ISENBERG*
*Bloomsbury Rheumatology Unit, Department of Rheumatology Research, University College and Middlesex School of Medicine, London; **Eastman Dental Hospital, London; †University of Birmingham Medical School, Birmingham, UK In this study the concentration of the different subclasses of IgG in sera from patients with range of autoimmune rheumatic diseases (ARD) was detected by radial immunodiffusion. In the second part the IgG subclasses of autoantibodies that recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), Ro, La, Sm and RNP in patients with ARD were measured by enzyme-linked immunosorbent assay. We studied 15 patients with lupus, 20 patients each with primary and secondary Sjögren’s syndrome (SS) and 10 each with rheumatoid arthritis (RA), scleroderma and myositis. Twenty healthy controls were also measured. The serum concentration of IgG2 in ARD patients was generally reduced. In contrast, the concentrations of IgG1, IgG3 and IgG4 subclasses were normal or raised. A high degree of correspondence in the IgG1, IgG2 and IgG3 responses to dsDNA and ssDNA in SLE was found. Notable differences in the IgG1 anti-Ro and ssDNA responses compared to the other subclasses were seen in 1°and 2° SS. In addition, an unexpected high level of IgG4 antibodies to ssDNA in 1° SS (65%) and IgG4 antibodies to Sm/RNP in RA was observed.
a
Key Words: IgG subclasses Autoimmune rheumatic diseases Anti-DNA antibodies the blood, I~;t3~ and IgG3 lymphocytes predominate in the tonsil lymphocytes s. 6. The serum of patients with systemic autoimmune rheumatic diseases (ARD) is characterized by reactivity to a broad spectrum of nuclear and some cytoplasmic components. Among these antibodies, those binding to
Introduction Five human classes of immunoglobulins have been defined (IgG, IgM, IgA, IgD and IgE) by the antigenic variation in the structure of their heavy chains, and subsequently subclasses of IgG and IgA have been identified’,’. It is now established that the nine heavy chain isotypes are products of discrete constant region genes encoded in man within the heavy chain locus of chromosome 14 and each may be expressed with rearranged variable region genes to yield a single polypeptide chain 2. The IgG isotype is the most common immunoglobulin in sera from human beings and has four subclasses. They differ in the primary structure of the Fc in the heavy chain of immunoglobulin and are designated Igt~rl, IgG2, IgG3 and IgG4’. Each has different biological pra~perti~s 4, for example in their capacity to fix complement. It is unclear how antibody subclass is selected, but different antigens may be associated with one subclass the nature of immunogen is likely to play an important role4. On the other hand, the localization of the ~-l ph~cyt~ subset and/or selective stimulation of B-cell subset factors in the local environment could also be relevant. Thus, while IgGl- and IgG2-bearing lymphocytes predominate in
DNA, Sm, RNP, Ro, La, histones, poly(ADP-ribose) and RNA have been studied extensively’,’. Hypergammaglobulinaemia, which is a common phenomenon in ARD, especially in systemic lupus erythematosus (SLE) and Sj6gren’s syndrome (SS), is invariably due to an increase in IgG9,1O. However, quantification of the subclass of IgG in these diseases is not well established and their role in pathogenesis is uncertain. Several reports have described IgG subclass distribution in ARD. Different studies have described selective or non-selective deposits of particular IgG subclasses in the kidneys of with SLE ~~> ~~. Some authors have reported a high level of total Igt~ and IgG3 against self-components&dquo; 11-16 . Other studies have identified selective among subclasses to ’selfcomponents’, for example IgGI1 to double-stranded DNA (dsDNA) or IgG3 to Sm in SLE patients Moreover, the serum level of IGGI and IgG3 against dsDNA in SLE has shown a correlation with disease activity in general and renal involvement in particular. However, the level of IgG2 anti-dsDNA antibodies correlated with joint and skin disease alone and in those with thrombotic episodes and spontaneous abortion &dquo;’. One report described a patient with SS with a selective
Correspondence: Professor D.A. Isenberg, M.D., F.R.C.P., Bloomsbury Rheumatology Unit, Arthur Stanley House, Tottenham Street, London WIP 9PG, UK.
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antibody deficiency of IgG2 and IgG4 as measured by radial immunodiffusion. This patient had recurrent pneumococcal bacteriaemia 20. This observation has been confirmed by other investigators who measured antibodies to a specific antigen (DNA)21.22. However, patients with SS immunized with tetanus toxoid (specific antigen response) were shown to have an Igt~I and IgG4 response similar to that of normal individuaIS21. In contrast, SLE patients have been reported to have a subnormal response of all the IgG subclasses against
toxoid 23. Other studies with nephelometry have described a selective increase in the concentration of IgG1 in the early stages of SS, and an inverse relation to the duration of the disease has been reported 9.10. Several groups of investigators have published tables of values for the normal ranges of IgG subclasses. Some studies have been done in children and others in adults 24-26 . However, there is a lack of concordance because of differences in reagents and assay protocols used. To our knowledge no study has reported the concentration of different IgG subclasses across the spectrum of ARD. The present study had two aims. The first was to determine the concentration of different subclasses of IgG in sera from patients with a range of ARD by radial immunodiffusion to ascertain differences in their distribution. The second was to characterize by enzymelinked immunosorbent assay (ELISA) the IgG subclass of antibodies recognizing a variety of relevant autoantigens, single-stranded DNA (ssDNA), dsDNA, Ro, La, Sm and RNP, across the range of ARD. tetanus
’
Materials and methods Serum
taken at random from stored sets of samples sera (stored at -2t~°~~ from patients who have attended the Bloomsbury Rheumatology Unit for long-term follow-up in the past 5 years. Thus, 20 sera from patients with primary and 20 with secondary SS, 15 patients with SLE and 10 patients each with rheumatoid arthritis (RA), myositis and scleroderma were used. All the patients with secondary SS also had SLE. Twenty healthy controls were used to establish a normal range. Test
were
Criteria for inclusion Each of the patients with SS was diagnosed by the criteria of Fax et al.21; we considered as primary those patients without any disease association, and as secondary those whose syndrome showed association with SLE only. The lupus patients met four or more of the American Rheumatism Association’s (ARA)
revised criteria for the classification of the disease 21 The patients with RA each met four or more of the revised ARA criteria 29. The patients with SLE were regularly assessed and only those without any evidence of SS in over 5 years’ follow-up were included in this group. The scleroderma patients all met the preliminary criteria of the ARA 30. The patients with myositis had three or more of the criteria described by Bohan and Peter31. The normal controls were individuals attending rheumatology out-patient clinics with soft tissue problems only; they did not have any clinical manifestations of autoimmune disease when sampled (15 female and 5 male, mean age 27 years). Having determined the level of each IgG subclass in the sera of the patients with SLE and 2° SS, we stratified the patients according to the amount of steroid prescribed (no steroid, less than 10mg/day or more than 10 mg/day) with or without azathioprine, in order to determine the effect of treatment on the level of immunoglobulin.
Antigens was obtained from Sigma Laboratories (St Louis, Mo., USA); ssDNA was prepared by the dsDNA solution being boiled for 15 min and then cooled on ice for 10 min. This preparation was sonicated for 5 min 32. Ro and La were prepared as part of the Shield diagnostic kit (Diastat kit, Shield, Dundee, UK). Sm/RNP was obtained from Imperial Laboratories Ltd (Andover, UK). In order to test the specificity of the polyclonal antibodies to IgG subclasses we tested each of the sheep anti-human monospecific to IgG 1, IgG2, IgG3 and IgG4 conjugated with alkaline phosphatase against IgG monoclonal antibodies to DNA at dilutions from
dsDNA
l:lfl(~ to 1:2 to find the best dilution of each conjugate (IgGI 1:1000, IgG2 1:1500, IgG3 1:200 and IgG4 1:200). We did not find any cross-reactivity between our reagents. Using different positive SLE sera against DNA we selected the positive controls for each IgG subclass.
Radial
immunodiffusion
A commercial kit (The Binding Site, Birmingham, UK) was used. This consisted of agar plates diluted with monospecific sera against IgG, IgG2, IgG3 and IgG4. The kit incorporates positive and negative controls. All the serum samples were diluted 1:10 in bovine serum albumin (BSA) 7% and 5 Al was added to each well. Incubation was performed in humid conditions at room temperature for 72 h. During this time the equilibrium between antigen and antibody was established. The precipitate ring diameter was accurately measured by immunodiffusion radial measurement. The
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the assay was completed as for the DNA antibody ELISA. Anti-Sm/RNP antibodies were measured by ELISA. Half the plate was coated with 2 JJ.g/ml of Sm/RNP diluted in bicarbonate buffer, pH 9.6, while the other half of the plate was incubated with bicarbonate buffer alone. This was done for all plates; the sera were diluted 1:200 and added in duplicate to the plates, which were then left for 1 h at ~?°~. The conjugate and the substrate were added with the same concentration as described above.
concentration was established with a reference table. The results were expressed in gll, The normal range was determined from the values obtained in the healthy individuals. ELISA Anti-ssDNA and anti-dsDNA antibodies were measured by ELISA. All reagents were added in 100-ILI volumes per well. Ninety-six-well flat-bottomed polystyrene microtitre plates (Nunc Maxisorp plates, Gibco Life Sciences, Uxbridge, UK) were coated with 50 jig/ml of POlY-L-lysine (Sigma Laboratories, Poole, UK) in phosphate buffered saline (PBS), pH 7.2, for 1 h at 37°~. Plates were washed three times with PBS and half the plate was coated with 5 ~cgl~ni of dsDNA (Sigma Laboratories) and ssDNA (10 tLg/ml), while the other half of the plate was incubated with PBS alone. This was done for all plates, which were then left overnight at 4°C. They were then washed with PBS and blocked with poly-L-glutamine/PBS for 1 h at 3’7°C. Then casein 2Vo in PBS was added for 1 h at 3~°C. Test and control sera were diluted 1/100 in PI~S-~1.5~1~ Tween (PBS-T) (Sigma Laboratories) for dsDNA and ssDNA and added in duplicate to the plates, which were then left for 1 h at 37°C. These were then washed three times with PBS-T and incubated with sheep anti-human monospecific to
Statistics For radial immunodiffusion the test values were considered raised if the mean diameter value was > + 2SD that observed with normal sera, and reduced if the mean diameter measurement ~as ~ - 2SD of the control value. For the ELISAs the test values were mean -~ ZSI3 considered positive if they had an OD > of the mean OD value of normal healthy control values, which were used to establish a normal range for the assay. Once the means and standard deviations of the tested groups were determined, the values were compared by Student’s t-test by means of the computational Exstatix 1.03 program on an Apple Macintosh
computer.
IgGl (1:1000 dilution), IgG2 (1:1500 dilution), IgG3
~1:2~U dilution) and IgG4 ~l:2tl~ dilution) conjugated with alkaline phosphatase (The Binding Site) for 1 h at 37°C. Unbound conjugate was removed by washing the plates with PBS-T and the substrate, dinitrophenyl phosphate (Sigma Laboratories), in bicarbonate buffer, pH 9.6, added at 1 n~~/rnl. The plates were then left at room temperature for 20 min before being read with an automated ELISA plate reader (Dynatech MR4000). Optical densities (OD) were measured at 405 nm. Final OD values were determined by the mean of duplicate readings obtained in the uncoated half of the plate being subtracted from the corresponding mean values obtained when the plates were coated with antigen. All the plates were run with the same positive and negative sera control to ensure the results were comparable. Anti-Ro and anti-La antibodies were measured by modification of an ELISA kit. We used the plates provided by Shield Diagnostic (Diastat kit) that were previously coated with Ro and La antigen and blocked. We added the test sera and controls in duplicate. The sera samples were diluted 1/100 in PBS-T, and then left for 2 h at room temperature. Then the plates were washed four times with PBS-T and incubated with sheep anti-human monospecific to Ig;~l (1:1000 dilution), IgG2 (1:1500 dilution), IgG3 (1:200 dilution) and IgG4 ~l:2t1~3 dilution) conjugated with alkaline phosphatase (The Binding Site) for 1 h at 37°C. The remainder of
Results
Figure 1 shows the individual serum levels of IgGl. The range for the normal controls was 5-11 gll (mean 8.26 ::i: 3.16). Only one normal person had a raised level. In contrast, raised levels of IgGl were found in 55% (I 1 c~f 20) of l ° SS and 50% (10 of 20) of 20 SS patients. Of the lupus and myositis patients, 30% (3 of 10) had high levels. Ten percent of the patients with sci~rc~d~rrr~a and RA had raised levels of this immunoglobulin subclass. In general, the highest levels of IgGl were seen in 1 and 2° SS and SLE with means of 12. l, 12.4 and 10.2 g/1, respectively. The values of these three groups were statistically significantly increased compared to the normals ~T~ ~ 0.001). The serum levels of IgG2 were normal or low, especially in those with I° ~~. The patients with 2° SS and SLE also had low levels of and the mean values of all three groups were statistically significantly reduced (P < 0.001) compared to the normal controls. However, we did not find any statistically significant differences between I and 2° SS (Figure 2). The IgG subclass values of the patients with RA, myositis and scleroderma were in the normal range without any statistically significant differences compared to the healthy group. Thirty-five percent of the lOSS patients 393
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Figure 1 line is the
Concentration of IgGI in serum from different ARD patients. Each mean and one standard deviation found in each disease.
Figure 2 Concentration of IgG2 in serum from different ARD patients. line is the
mean
and
one
Each
point shows the value of each serum sample tested. The horizontal
point shows the value of each serum sample tested. The horizontal
standard deviation found in each disease.
showed a deficiency of and 45% of the patients with 2° SS had a similar deficiency. In contrast, 15% of the SLE patients had a deficiency of this immunoglobulin. Ten percent of the with myositis and RA had reduced values. IgG3 levels (Figure 3) were commonly raised, although less frequently and of less magnitude compared to IGGI levels. A raised level of IgG3 was often present in those patients with raised levels of IgG 1. Increased levels were present in 2~~~ of the 2° SS, 20% of the 1 SS, 40% of the SLE, 30% of the RA and 10% of the scleroderma patients. There was no relationship between those patients who had raised IgC~1 and IgG3
levels and those with low levels. A raised level of IgG4 was found in two patients with myositis (Figure 4) and two SLE patients, although the latter levels were only slightly raised. The rest of the groups studied were in the normal range. There were no statistically significant differences in any of the patient groups, compared to the normal controls. In order to establish whether the treatment of the patients had any effect on the level of immunoglobulin, we stratified the SLE and 2° SS patients according to the corticosteroids and azathioprine they were taking (see methods). The patients on more than 10 mg of steroid plus azathioprine had lower levels of IgGI and
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Figure 3 line is the
Concentration of IgG3 in serum from different ARD patients. Each mean and one standard deviation found in each disease.
point shows the value of each serum sample tested. The horizontal
Figure 4 Concentration of lgG4 in serum from different ARD patients. Each point shows the value of each serum line is the mean and one standard deviation found in each disease.
sample tested. The horizontal
anti-dsDNA antibodies. Primary SS had a similar distribution to the with 2° SS: 25% of antibodies dsDNA were IgG4, and 5% were and IgG3. Antibodies dsDNA were not detected in patients with other ARD, apart from one patient with scieroderma who had a raised IgG2 level. Antibodies against ssDNA were common in those ARD patients with manifestations of ’non-organspecific autoimmune phenomena’. The distribution was interesting: 66% of the SLE patients had IgGi to ssDNA, with raised levels of IgG2 in 40%, IgG3 in 26% and IgG4 in 6%. !gGl and IgG4 to ssDNA in
IgG3 immunoglobulin (range 5-10 g/1). In contrast, those on less than 10 mg or those without any treatment showed values in all the ranges. Table I shows the frequency of patients with a raised level of IgG subclasses against ~isI~I~I~. and ~sl~N~i. SLE patients had a predominant restriction of IgGI to dsDNA ~4~~~a~, especially in those patients with grade 4 activity (data not shown). Raised levels of IgG2, IgG3 and IgG4 were detected at a lower frequency ~~~~~, 20% and 6%, respectively). In contrast, the isotype of antibodies to dsDNA in patients with 2° SS was IgG4 (45Vo); only 10% of 2° SS patients had IGGI 395
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Table I
patients.
IgG subclasses of autoantibodies to dsDNA and ssDNA in serum from ARD The table shows the frequency of positive for each IgG subclass in each group of
disease studied.
1° and 2° SS: primary and RA: rheumatoid arthritis.
secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus;
IgG subclasses of autoantibodies to La and Ro in serum from ARD patients. The table shows the frequency of positive for each IgG subclass in 1 and 2° SS and SLE. Table ~T
1° and 2° SS:
primary and secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus.
1 and 2° SS were inversely proportional to the antidsDNA subclass levels. Thus, patients with 14 SS and 2° SS had raised IgG4 levels to ssDNA in ~5 ~~ and SS~I~, respectively. The frequency of IgG2 and IgG3 was low in both 1 and 2° SS. Fifteen percent of scieroderma patients had raised Ig(~1 anti-ssl~N~ levels; IgG2 was detected in 5% and IgG3 in 10%. Anti-La and anti-Ro antibodies (Table II) were tested only in the patients with lupus and SS. Only one SLE patient had antibodies to La of the Ig~l and IgG2 subclasses. The frequency of detection in IgG subclasses detected against La in 1 and 2° SS was similar. Raised IgG subclasses were restricted to IgGI 25%, IgG2 30% and IgG3 15070 in both groups, and in only one case was a high IgG4 level in 2‘~ SS found. In contrast to anti-La antibodies, 70Vo of 1° SS patients had raised levels of IgCil to Ro, but only 5~~ had raised IgG2 and 10% IgG3 levels. Secondary SS showed a similar restriction, with 55% of patients having increased IgC:~I levels and only 10% IgG2 and 5o IgG3. Two lupus patients without SS had antibodies to Ro, the subclass distribution being Ig(~I l 1 3 .3 Vo , IgG2 6.6% and IgG3 6.6so . Analysis of the IgG subclasses to Sm/RNP (Table III) shows that five of the 12 SLE patients (2° SS and SLE groups) tested (I ~.6~,a~ had antibodies to Sm/RNP of the IgGI types, although four of these also had SS. Two patients in the 1° SS group had IgG1, 2 and 3 anti-Sm/
’I’~~1~ III IgG subclasses of autoantibodies to Sm/RNP in serum from ARD patients. The table shows the frequency of positive for each IgG subclass in each group studied.
1° and 2° SS: primary and secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus; RA: rheumatoid arthritis.
RNP antibodies. Among the scleroderma patients raised levels of IgGI antibodies to Sm/RNP were present in 2~~~ and IgG3 in 10%. IgG2 and IgG4 levels were not increased. RA patients in contrast showed elevated IgG4 anti-Sm/RNP levels in 3(3~I~ of those studied.
Discussion In this study we have reported on the IgG subclass levels in patients with a range of ARD. Although SLE and SS are both well known to have an accompanying hypergammaglobulinaemia, this has also been reported in both polymyositis I dermatomyositis 33 and
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scleroderma 34. The presence of particular IgG subclasses in patients’ sera has implications for the nature
We found autoantibody activity in all four IgG subclasses, with the proportion of anti-’self-component’
of the
in each subclass varying greatly in different individuals. We did not find IgG subclass antibodies to dsDNA and ssDNA in those ARD generally regarded as organ specific, e.g. myositis. This observation supports the view that antibodies to DNA are restricted to ’nonorgan-specific disease’ and that IGGI and IgG4 are found preferentially. IgG subclass antibodies against La and Ro were tested only in the SLE and SS patients. The pattern recorded was similar in the patients with 1 and 2° SS. Raised levels of IgGl, IgG2 and IgG3 against La and a selective restriction of IgGI1 to Ro were present in both types of SS. A few SLE patients had antibodies- to La and Ro, mostly of the Ig~l and IgG2
antigen that
may have
triggered the response. particularly associated with the immune response to proteins 35. In contrast, IgG2 is more frequently seen after immunization with polysaccharides 36, 37 and IgG4 has frequently been reported in the sera of patients with hypersensitivities, haemophilias with antibodies to VIII or IX factor, and antithyroglobulin and antimicrosome antibodies in Hashimoto’s disease 38-40. The deficiency of any particular IgG subclass may predispose to infectious disease. For example, IgG2 deficiency has been related to Haemophilus ~rt,f~~a~~.~t~e type b and pneumococcal infections&dquo;. IgG3 deficiency, in contrast, has been related to a history of recurrent upper and lower respiratory tract infection and bronchial asthma 42. Our results indicate that patients with ARD often have high serum levels of IgG 1, IgG3 and IgG4 and that IgG and IgG3 levels are often elevated in parallel. Raised levels were most often present in patients with SLE, 2° SS and 1 ~~‘, in contrast to patients with RA, myositis and scleroderma. Raised levels of IgG4 were found in only two patients with lupus, but were also high in two other patients with myositis, perhaps suggesting that they had been exposed to chronic antigenic stimulation with protein antigens 43. Some ARD patients had a low level of IgG2. This is in agreement with a previous report of these conditions 44. It was notable that nearly 50% of the patients Thus, ~~C.~l and IgG3
are
isotypes. The pattern of antibodies to Sm/RNP was more ’promiscuous’. was the most common subclass to Sm/RNP in 2° SS and scl~ruder~na. In contrast, raised IgG2 levels were recorded only in I ° SS (I(1~7o) and SLE (8.3%), whereas IgG3 and/or IgG4 against Sm/RNP were present in all of the diseases (IgG4 in 30% of the RA patients). We did not find a correlation between the concentration of IgG subclasses in the sera and recognition of ‘s~elf-~c~~np~n~nts’. Perhaps the high level of IgG in ARD patients with hypergammaglobulinaemia is due to non-specific activation of B cells stimulating multiple clones not all of which are directed against ’selfcomponents’. The high levels of IgGl and IgG3 may be due to the related close genetic linkage of the ’Y1 and y3 genes, implying that stimulation caused a sequential switching among the genes coding for the constant region of the heavy chain within the B-cell genome. Alternatively, the pattern of the Ig~&dquo;r subclasses against ’self-component’ could be due to a polymorphic antigen (such as a super antigen) that triggered this response and the persistence of this antigen could explain the
with both 1 and 20 SS had reduced levels. In contrast, only 10-15% of the patients with SLE, myositis and RA had evidence of IgG2 deficiency. In the SLE patients the levels of IgG subclasses have been reported to show a correlation with susceptibility to infectious disease, notably low levels of IgG3 and IgG4, which are linked to mycotic infection. Oxelius et al.45 found that IgG2 and IgG4 deficiencies were associated with IgA deficiency. Other authors, in contrast, have reported a correlation between disease activity (in SLE) and an increase of IgG antibodies to tetanus and cytomegalovirus&dquo;. Our results show that ARD patients, especially those with SS SLE, have a selective IgG2 deficiency and thus they may be more susceptible to infectious disease than the normal person~. We stratified the SLE and 2° SS patients according to their corticosteroid and azathioprine requirements. Because our sample size is relatively small we are cautious about interpreting the data, but the patients on more than 10 mg of steroid plus azathioprine had low levels of I~~l and IgG3 immunoglobulin compared with those on less than 10 mg or those without any treatment. Large numbers of patients will need to be studied to consider the possible effects of the treatment and disease activity.
high level of IgG4. These results may be compared to the relatively few studies of IgG subclass distribution in patients with infectious disease. For example, Zouali et al.’ showed that in patients with acute malaria 18070 had antibodies to dsDNA and that these wore of the IgGl subclass, while the antibodies to ssDNA (22%) and RNP (32Vo) were more evenly spread the subclasses, especially in the former case. In contrast, IgG2 was found to be the predominant subclass among antibodies binding a .~~yct~~c~ct~ri~~ leprae sonicate extract in the sera of patients with leprosy, although lgd~rl and IgG4 were more prominent in certain disease
subtypes 48
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Acknowledgements This work was supported in part by Programa Universitario de Investi~aeic~n en Salud (PUIS-DGAPA) from the Autonomous National University of M6xico (UNAM), and by the Arthritis and Rheumatism
Council, UK.
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(abstract D29).
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22. Zouali M, Jefferris R, Eyquem A. IgG subclass distribution of autoantibodies to DNA and to nuclear ribonucleoproteins in autoimmune disease. Immunology 1984; 51: 595-600. 23. Rubin RL, Tang FL, Lucas AH, Spiegelberg HL, Tan EM. IgG subclasses of anti-tetanus toxoid antibodies in adult and newborn normal subjects and patients with systemic lupus erythematosus, Sjögren’s syndrome, and drug-induced autoImmunol 137: 2522-7. immunity. J 1986; 24. Oxelius VA. Quantitative and qualitative investigation of serum IgG subclasses in immunodeficiency disease. Clin Exp Immunol 1979; 36: 112-6. 25. Morell A, Skvaril F, Steinberg AG, Van Loghem E, Terry WD. Correlations between the concentrations of the four subclasses of IgG and Gm allotypes of normal human sera. J Immunol 1972; 108: 195-206. 26. Van Der Giessen M, Roussouw E, Algra-Van Veen T, Van-Loghem E, Zegers BJM, Sander PC. Quantitation of IgG subclasses in sera of normal adults and healthy children between 4 and 12 years of age. Clin Exp Immunol 1975; 21: 501-9. 27. Fox RI, Robinson ChA, Curd JG, Kozin F, Howell FV. Sjögren’s syndrome: proposed criteria for classification. Arthritis Rheum 1986; 29: 577-85. 28. Tan EM, Cohen AJ, Fries et JF al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271-7. 29. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: 315-24. 30. Masi AT, Rodnan GP, Medsger RA. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Bull Rheum Dis 1981; 31: 1-6. 31. Bohan A, Peter JB. Polymyositis and dermatomyositis. New Engl J Med 1975; 292: 344-7. 32. Le Page S, Williams W, Parkhouse D et al. Relationship between lymphocytotoxic antibodies, anti-DNA antibodies, and a common anti-DNA idiotype PR4 in patients with SLE, their relatives and spouses. Clin Exp Immunol 1989; 77: 314-8. 33. Lisak RP, Zweimann B. Serum immunoglobulin level in myasthenia gravis, polymyositis and dermatomyositis. J Neurol Neurosurg Psychiat 1976; 39: 34-7. 34. Schumaker NR, ed. Primer in Rheumatic Disease, 9th edn. Atlanta: Arthritis Foundations, 1988: 115. 35. Bankhurst AD, Lamber PH, Miescher PA. Studies on the thymic dependence of the immunoglobulin classes of the mouse. Proc Soc Exp Biol Med 1975; 148: 501-4. 36. Yount WJ, Dorner MM, Hunkel HG, Habat EA. Studies on human antibodies. VI. Selective variations in subgroups composition and genetic markers. J Exp Med 1968; 127 : 633-46. 37. Frejid A, Hammarstrom L, Persson MAA, Smith CIE. Plasma anti-pneumococcal antibody activity of the IgG class and subclasses in otitis prone children. Clin Exp Immunol 1984; 56: 233-8. 38. Merret J, Barnetson RSTC, Burr ML, Merret TG. Total and specific IgG4 antibody level in atopic eczema. Clin Exp Immunol : 645-52. 1984; 56 39. Pike IM, Yount WJ, Puritz EM, Roberts HR. Immunochemical characterisation of monoclonal γG4, λ human antibody to factor IX. Blood 1972; 40: 1-10. 40. Parkes AB, McLachlan SM, Bird P, Rees Smith B. The distribution of microsomal and thyroglobulin antibody activity among the IgG subclasses. Clin Exp Immunol 1984; 57: 239-43. 41. Goldblatt D, Morgan G, Seymour ND, Trobel S, Turner MW, Levinsky RJ. The clinical manifestation of IgG subclass defi-
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In: Levinsky RJ, ed. IgG Subclass Deficiencies. London: Royal Society of Medicine, 1989: 19. 42. Oxelius VA, Hanson LA, Bjorkander J, Hammarstrom L,
45. Oxelius VA. Immunoglobulin G (IgG) subclasses and human disease. Am J Med 1984; 76: 7-18. 46. Jonsson H, Nived O, Sturfelt G. Outcome in systemic lupus
Sjoholm A. IgG3 deficiency: common in obstructive lung disease. In: Hanson LA, Soderstrom T, Oxelius VA, eds. Immunoglobulin Subclass Deficiency. Basel: Karger, 1986: 106. 43. Jefferis R, Kumararatne DS. Selective IgG subclass deficiency: quantification and clinical relevance. Clin Exp Immunol 1990;
erythematosus: a prospective study of patients from a defined population. Medicine (Baltimore) 1989; 68: 141-50. 47. Zouali M, Druilhe P, Eyquem A. IgG-subclass expression of
ciency.
anti-DNA and anti-ribonucleoprotein autoantibodies in human malaria. Clin Exp Immunol 1986; 66: 273-8. 48. Dhandayuthapani S, Izumi S, Anandan D, Bhatia VN. Specificity of IgG subclass antibodies in different clinical manifestations of leprosy. Clin Exp Immunol 1992; 88: 253-7.
81: 357-67. 44. Tokano Y, Yagita H, Iida N, Hashimoto H, Okumura K, Hirosone S. Relation between the level of IgG subclasses and infections in patients with systemic lupus erythematosus. Int Arch Allergy Appl Immunol 1988; 87: 55-8.
-
(Received 9 August 1992) 17 September 1992)
(Accepted
399
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Systemic
Lupus Erythematosus and Other
Autoimmune Rheumatic Diseases F.
BLANCO*,
J.
KALSI*,
C.T.
RAVIRAJAN*,
P.
SPEIGHT**,
A.R. BRADWELL † and D.A. ISENBERG*
*Bloomsbury Rheumatology Unit, Department of Rheumatology Research, University College and Middlesex School of Medicine, London; **Eastman Dental Hospital, London; †University of Birmingham Medical School, Birmingham, UK In this study the concentration of the different subclasses of IgG in sera from patients with range of autoimmune rheumatic diseases (ARD) was detected by radial immunodiffusion. In the second part the IgG subclasses of autoantibodies that recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), Ro, La, Sm and RNP in patients with ARD were measured by enzyme-linked immunosorbent assay. We studied 15 patients with lupus, 20 patients each with primary and secondary Sjögren’s syndrome (SS) and 10 each with rheumatoid arthritis (RA), scleroderma and myositis. Twenty healthy controls were also measured. The serum concentration of IgG2 in ARD patients was generally reduced. In contrast, the concentrations of IgG1, IgG3 and IgG4 subclasses were normal or raised. A high degree of correspondence in the IgG1, IgG2 and IgG3 responses to dsDNA and ssDNA in SLE was found. Notable differences in the IgG1 anti-Ro and ssDNA responses compared to the other subclasses were seen in 1°and 2° SS. In addition, an unexpected high level of IgG4 antibodies to ssDNA in 1° SS (65%) and IgG4 antibodies to Sm/RNP in RA was observed.
a
Key Words: IgG subclasses Autoimmune rheumatic diseases Anti-DNA antibodies the blood, I~;t3~ and IgG3 lymphocytes predominate in the tonsil lymphocytes s. 6. The serum of patients with systemic autoimmune rheumatic diseases (ARD) is characterized by reactivity to a broad spectrum of nuclear and some cytoplasmic components. Among these antibodies, those binding to
Introduction Five human classes of immunoglobulins have been defined (IgG, IgM, IgA, IgD and IgE) by the antigenic variation in the structure of their heavy chains, and subsequently subclasses of IgG and IgA have been identified’,’. It is now established that the nine heavy chain isotypes are products of discrete constant region genes encoded in man within the heavy chain locus of chromosome 14 and each may be expressed with rearranged variable region genes to yield a single polypeptide chain 2. The IgG isotype is the most common immunoglobulin in sera from human beings and has four subclasses. They differ in the primary structure of the Fc in the heavy chain of immunoglobulin and are designated Igt~rl, IgG2, IgG3 and IgG4’. Each has different biological pra~perti~s 4, for example in their capacity to fix complement. It is unclear how antibody subclass is selected, but different antigens may be associated with one subclass the nature of immunogen is likely to play an important role4. On the other hand, the localization of the ~-l ph~cyt~ subset and/or selective stimulation of B-cell subset factors in the local environment could also be relevant. Thus, while IgGl- and IgG2-bearing lymphocytes predominate in
DNA, Sm, RNP, Ro, La, histones, poly(ADP-ribose) and RNA have been studied extensively’,’. Hypergammaglobulinaemia, which is a common phenomenon in ARD, especially in systemic lupus erythematosus (SLE) and Sj6gren’s syndrome (SS), is invariably due to an increase in IgG9,1O. However, quantification of the subclass of IgG in these diseases is not well established and their role in pathogenesis is uncertain. Several reports have described IgG subclass distribution in ARD. Different studies have described selective or non-selective deposits of particular IgG subclasses in the kidneys of with SLE ~~> ~~. Some authors have reported a high level of total Igt~ and IgG3 against self-components&dquo; 11-16 . Other studies have identified selective among subclasses to ’selfcomponents’, for example IgGI1 to double-stranded DNA (dsDNA) or IgG3 to Sm in SLE patients Moreover, the serum level of IGGI and IgG3 against dsDNA in SLE has shown a correlation with disease activity in general and renal involvement in particular. However, the level of IgG2 anti-dsDNA antibodies correlated with joint and skin disease alone and in those with thrombotic episodes and spontaneous abortion &dquo;’. One report described a patient with SS with a selective
Correspondence: Professor D.A. Isenberg, M.D., F.R.C.P., Bloomsbury Rheumatology Unit, Arthur Stanley House, Tottenham Street, London WIP 9PG, UK.
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antibody deficiency of IgG2 and IgG4 as measured by radial immunodiffusion. This patient had recurrent pneumococcal bacteriaemia 20. This observation has been confirmed by other investigators who measured antibodies to a specific antigen (DNA)21.22. However, patients with SS immunized with tetanus toxoid (specific antigen response) were shown to have an Igt~I and IgG4 response similar to that of normal individuaIS21. In contrast, SLE patients have been reported to have a subnormal response of all the IgG subclasses against
toxoid 23. Other studies with nephelometry have described a selective increase in the concentration of IgG1 in the early stages of SS, and an inverse relation to the duration of the disease has been reported 9.10. Several groups of investigators have published tables of values for the normal ranges of IgG subclasses. Some studies have been done in children and others in adults 24-26 . However, there is a lack of concordance because of differences in reagents and assay protocols used. To our knowledge no study has reported the concentration of different IgG subclasses across the spectrum of ARD. The present study had two aims. The first was to determine the concentration of different subclasses of IgG in sera from patients with a range of ARD by radial immunodiffusion to ascertain differences in their distribution. The second was to characterize by enzymelinked immunosorbent assay (ELISA) the IgG subclass of antibodies recognizing a variety of relevant autoantigens, single-stranded DNA (ssDNA), dsDNA, Ro, La, Sm and RNP, across the range of ARD. tetanus
’
Materials and methods Serum
taken at random from stored sets of samples sera (stored at -2t~°~~ from patients who have attended the Bloomsbury Rheumatology Unit for long-term follow-up in the past 5 years. Thus, 20 sera from patients with primary and 20 with secondary SS, 15 patients with SLE and 10 patients each with rheumatoid arthritis (RA), myositis and scleroderma were used. All the patients with secondary SS also had SLE. Twenty healthy controls were used to establish a normal range. Test
were
Criteria for inclusion Each of the patients with SS was diagnosed by the criteria of Fax et al.21; we considered as primary those patients without any disease association, and as secondary those whose syndrome showed association with SLE only. The lupus patients met four or more of the American Rheumatism Association’s (ARA)
revised criteria for the classification of the disease 21 The patients with RA each met four or more of the revised ARA criteria 29. The patients with SLE were regularly assessed and only those without any evidence of SS in over 5 years’ follow-up were included in this group. The scleroderma patients all met the preliminary criteria of the ARA 30. The patients with myositis had three or more of the criteria described by Bohan and Peter31. The normal controls were individuals attending rheumatology out-patient clinics with soft tissue problems only; they did not have any clinical manifestations of autoimmune disease when sampled (15 female and 5 male, mean age 27 years). Having determined the level of each IgG subclass in the sera of the patients with SLE and 2° SS, we stratified the patients according to the amount of steroid prescribed (no steroid, less than 10mg/day or more than 10 mg/day) with or without azathioprine, in order to determine the effect of treatment on the level of immunoglobulin.
Antigens was obtained from Sigma Laboratories (St Louis, Mo., USA); ssDNA was prepared by the dsDNA solution being boiled for 15 min and then cooled on ice for 10 min. This preparation was sonicated for 5 min 32. Ro and La were prepared as part of the Shield diagnostic kit (Diastat kit, Shield, Dundee, UK). Sm/RNP was obtained from Imperial Laboratories Ltd (Andover, UK). In order to test the specificity of the polyclonal antibodies to IgG subclasses we tested each of the sheep anti-human monospecific to IgG 1, IgG2, IgG3 and IgG4 conjugated with alkaline phosphatase against IgG monoclonal antibodies to DNA at dilutions from
dsDNA
l:lfl(~ to 1:2 to find the best dilution of each conjugate (IgGI 1:1000, IgG2 1:1500, IgG3 1:200 and IgG4 1:200). We did not find any cross-reactivity between our reagents. Using different positive SLE sera against DNA we selected the positive controls for each IgG subclass.
Radial
immunodiffusion
A commercial kit (The Binding Site, Birmingham, UK) was used. This consisted of agar plates diluted with monospecific sera against IgG, IgG2, IgG3 and IgG4. The kit incorporates positive and negative controls. All the serum samples were diluted 1:10 in bovine serum albumin (BSA) 7% and 5 Al was added to each well. Incubation was performed in humid conditions at room temperature for 72 h. During this time the equilibrium between antigen and antibody was established. The precipitate ring diameter was accurately measured by immunodiffusion radial measurement. The
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the assay was completed as for the DNA antibody ELISA. Anti-Sm/RNP antibodies were measured by ELISA. Half the plate was coated with 2 JJ.g/ml of Sm/RNP diluted in bicarbonate buffer, pH 9.6, while the other half of the plate was incubated with bicarbonate buffer alone. This was done for all plates; the sera were diluted 1:200 and added in duplicate to the plates, which were then left for 1 h at ~?°~. The conjugate and the substrate were added with the same concentration as described above.
concentration was established with a reference table. The results were expressed in gll, The normal range was determined from the values obtained in the healthy individuals. ELISA Anti-ssDNA and anti-dsDNA antibodies were measured by ELISA. All reagents were added in 100-ILI volumes per well. Ninety-six-well flat-bottomed polystyrene microtitre plates (Nunc Maxisorp plates, Gibco Life Sciences, Uxbridge, UK) were coated with 50 jig/ml of POlY-L-lysine (Sigma Laboratories, Poole, UK) in phosphate buffered saline (PBS), pH 7.2, for 1 h at 37°~. Plates were washed three times with PBS and half the plate was coated with 5 ~cgl~ni of dsDNA (Sigma Laboratories) and ssDNA (10 tLg/ml), while the other half of the plate was incubated with PBS alone. This was done for all plates, which were then left overnight at 4°C. They were then washed with PBS and blocked with poly-L-glutamine/PBS for 1 h at 3’7°C. Then casein 2Vo in PBS was added for 1 h at 3~°C. Test and control sera were diluted 1/100 in PI~S-~1.5~1~ Tween (PBS-T) (Sigma Laboratories) for dsDNA and ssDNA and added in duplicate to the plates, which were then left for 1 h at 37°C. These were then washed three times with PBS-T and incubated with sheep anti-human monospecific to
Statistics For radial immunodiffusion the test values were considered raised if the mean diameter value was > + 2SD that observed with normal sera, and reduced if the mean diameter measurement ~as ~ - 2SD of the control value. For the ELISAs the test values were mean -~ ZSI3 considered positive if they had an OD > of the mean OD value of normal healthy control values, which were used to establish a normal range for the assay. Once the means and standard deviations of the tested groups were determined, the values were compared by Student’s t-test by means of the computational Exstatix 1.03 program on an Apple Macintosh
computer.
IgGl (1:1000 dilution), IgG2 (1:1500 dilution), IgG3
~1:2~U dilution) and IgG4 ~l:2tl~ dilution) conjugated with alkaline phosphatase (The Binding Site) for 1 h at 37°C. Unbound conjugate was removed by washing the plates with PBS-T and the substrate, dinitrophenyl phosphate (Sigma Laboratories), in bicarbonate buffer, pH 9.6, added at 1 n~~/rnl. The plates were then left at room temperature for 20 min before being read with an automated ELISA plate reader (Dynatech MR4000). Optical densities (OD) were measured at 405 nm. Final OD values were determined by the mean of duplicate readings obtained in the uncoated half of the plate being subtracted from the corresponding mean values obtained when the plates were coated with antigen. All the plates were run with the same positive and negative sera control to ensure the results were comparable. Anti-Ro and anti-La antibodies were measured by modification of an ELISA kit. We used the plates provided by Shield Diagnostic (Diastat kit) that were previously coated with Ro and La antigen and blocked. We added the test sera and controls in duplicate. The sera samples were diluted 1/100 in PBS-T, and then left for 2 h at room temperature. Then the plates were washed four times with PBS-T and incubated with sheep anti-human monospecific to Ig;~l (1:1000 dilution), IgG2 (1:1500 dilution), IgG3 (1:200 dilution) and IgG4 ~l:2t1~3 dilution) conjugated with alkaline phosphatase (The Binding Site) for 1 h at 37°C. The remainder of
Results
Figure 1 shows the individual serum levels of IgGl. The range for the normal controls was 5-11 gll (mean 8.26 ::i: 3.16). Only one normal person had a raised level. In contrast, raised levels of IgGl were found in 55% (I 1 c~f 20) of l ° SS and 50% (10 of 20) of 20 SS patients. Of the lupus and myositis patients, 30% (3 of 10) had high levels. Ten percent of the patients with sci~rc~d~rrr~a and RA had raised levels of this immunoglobulin subclass. In general, the highest levels of IgGl were seen in 1 and 2° SS and SLE with means of 12. l, 12.4 and 10.2 g/1, respectively. The values of these three groups were statistically significantly increased compared to the normals ~T~ ~ 0.001). The serum levels of IgG2 were normal or low, especially in those with I° ~~. The patients with 2° SS and SLE also had low levels of and the mean values of all three groups were statistically significantly reduced (P < 0.001) compared to the normal controls. However, we did not find any statistically significant differences between I and 2° SS (Figure 2). The IgG subclass values of the patients with RA, myositis and scleroderma were in the normal range without any statistically significant differences compared to the healthy group. Thirty-five percent of the lOSS patients 393
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Figure 1 line is the
Concentration of IgGI in serum from different ARD patients. Each mean and one standard deviation found in each disease.
Figure 2 Concentration of IgG2 in serum from different ARD patients. line is the
mean
and
one
Each
point shows the value of each serum sample tested. The horizontal
point shows the value of each serum sample tested. The horizontal
standard deviation found in each disease.
showed a deficiency of and 45% of the patients with 2° SS had a similar deficiency. In contrast, 15% of the SLE patients had a deficiency of this immunoglobulin. Ten percent of the with myositis and RA had reduced values. IgG3 levels (Figure 3) were commonly raised, although less frequently and of less magnitude compared to IGGI levels. A raised level of IgG3 was often present in those patients with raised levels of IgG 1. Increased levels were present in 2~~~ of the 2° SS, 20% of the 1 SS, 40% of the SLE, 30% of the RA and 10% of the scleroderma patients. There was no relationship between those patients who had raised IgC~1 and IgG3
levels and those with low levels. A raised level of IgG4 was found in two patients with myositis (Figure 4) and two SLE patients, although the latter levels were only slightly raised. The rest of the groups studied were in the normal range. There were no statistically significant differences in any of the patient groups, compared to the normal controls. In order to establish whether the treatment of the patients had any effect on the level of immunoglobulin, we stratified the SLE and 2° SS patients according to the corticosteroids and azathioprine they were taking (see methods). The patients on more than 10 mg of steroid plus azathioprine had lower levels of IgGI and
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Figure 3 line is the
Concentration of IgG3 in serum from different ARD patients. Each mean and one standard deviation found in each disease.
point shows the value of each serum sample tested. The horizontal
Figure 4 Concentration of lgG4 in serum from different ARD patients. Each point shows the value of each serum line is the mean and one standard deviation found in each disease.
sample tested. The horizontal
anti-dsDNA antibodies. Primary SS had a similar distribution to the with 2° SS: 25% of antibodies dsDNA were IgG4, and 5% were and IgG3. Antibodies dsDNA were not detected in patients with other ARD, apart from one patient with scieroderma who had a raised IgG2 level. Antibodies against ssDNA were common in those ARD patients with manifestations of ’non-organspecific autoimmune phenomena’. The distribution was interesting: 66% of the SLE patients had IgGi to ssDNA, with raised levels of IgG2 in 40%, IgG3 in 26% and IgG4 in 6%. !gGl and IgG4 to ssDNA in
IgG3 immunoglobulin (range 5-10 g/1). In contrast, those on less than 10 mg or those without any treatment showed values in all the ranges. Table I shows the frequency of patients with a raised level of IgG subclasses against ~isI~I~I~. and ~sl~N~i. SLE patients had a predominant restriction of IgGI to dsDNA ~4~~~a~, especially in those patients with grade 4 activity (data not shown). Raised levels of IgG2, IgG3 and IgG4 were detected at a lower frequency ~~~~~, 20% and 6%, respectively). In contrast, the isotype of antibodies to dsDNA in patients with 2° SS was IgG4 (45Vo); only 10% of 2° SS patients had IGGI 395
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Table I
patients.
IgG subclasses of autoantibodies to dsDNA and ssDNA in serum from ARD The table shows the frequency of positive for each IgG subclass in each group of
disease studied.
1° and 2° SS: primary and RA: rheumatoid arthritis.
secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus;
IgG subclasses of autoantibodies to La and Ro in serum from ARD patients. The table shows the frequency of positive for each IgG subclass in 1 and 2° SS and SLE. Table ~T
1° and 2° SS:
primary and secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus.
1 and 2° SS were inversely proportional to the antidsDNA subclass levels. Thus, patients with 14 SS and 2° SS had raised IgG4 levels to ssDNA in ~5 ~~ and SS~I~, respectively. The frequency of IgG2 and IgG3 was low in both 1 and 2° SS. Fifteen percent of scieroderma patients had raised Ig(~1 anti-ssl~N~ levels; IgG2 was detected in 5% and IgG3 in 10%. Anti-La and anti-Ro antibodies (Table II) were tested only in the patients with lupus and SS. Only one SLE patient had antibodies to La of the Ig~l and IgG2 subclasses. The frequency of detection in IgG subclasses detected against La in 1 and 2° SS was similar. Raised IgG subclasses were restricted to IgGI 25%, IgG2 30% and IgG3 15070 in both groups, and in only one case was a high IgG4 level in 2‘~ SS found. In contrast to anti-La antibodies, 70Vo of 1° SS patients had raised levels of IgCil to Ro, but only 5~~ had raised IgG2 and 10% IgG3 levels. Secondary SS showed a similar restriction, with 55% of patients having increased IgC:~I levels and only 10% IgG2 and 5o IgG3. Two lupus patients without SS had antibodies to Ro, the subclass distribution being Ig(~I l 1 3 .3 Vo , IgG2 6.6% and IgG3 6.6so . Analysis of the IgG subclasses to Sm/RNP (Table III) shows that five of the 12 SLE patients (2° SS and SLE groups) tested (I ~.6~,a~ had antibodies to Sm/RNP of the IgGI types, although four of these also had SS. Two patients in the 1° SS group had IgG1, 2 and 3 anti-Sm/
’I’~~1~ III IgG subclasses of autoantibodies to Sm/RNP in serum from ARD patients. The table shows the frequency of positive for each IgG subclass in each group studied.
1° and 2° SS: primary and secondary Sj6gren’s syndrome; SLE: systemic lupus erythematosus; RA: rheumatoid arthritis.
RNP antibodies. Among the scleroderma patients raised levels of IgGI antibodies to Sm/RNP were present in 2~~~ and IgG3 in 10%. IgG2 and IgG4 levels were not increased. RA patients in contrast showed elevated IgG4 anti-Sm/RNP levels in 3(3~I~ of those studied.
Discussion In this study we have reported on the IgG subclass levels in patients with a range of ARD. Although SLE and SS are both well known to have an accompanying hypergammaglobulinaemia, this has also been reported in both polymyositis I dermatomyositis 33 and
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scleroderma 34. The presence of particular IgG subclasses in patients’ sera has implications for the nature
We found autoantibody activity in all four IgG subclasses, with the proportion of anti-’self-component’
of the
in each subclass varying greatly in different individuals. We did not find IgG subclass antibodies to dsDNA and ssDNA in those ARD generally regarded as organ specific, e.g. myositis. This observation supports the view that antibodies to DNA are restricted to ’nonorgan-specific disease’ and that IGGI and IgG4 are found preferentially. IgG subclass antibodies against La and Ro were tested only in the SLE and SS patients. The pattern recorded was similar in the patients with 1 and 2° SS. Raised levels of IgGl, IgG2 and IgG3 against La and a selective restriction of IgGI1 to Ro were present in both types of SS. A few SLE patients had antibodies- to La and Ro, mostly of the Ig~l and IgG2
antigen that
may have
triggered the response. particularly associated with the immune response to proteins 35. In contrast, IgG2 is more frequently seen after immunization with polysaccharides 36, 37 and IgG4 has frequently been reported in the sera of patients with hypersensitivities, haemophilias with antibodies to VIII or IX factor, and antithyroglobulin and antimicrosome antibodies in Hashimoto’s disease 38-40. The deficiency of any particular IgG subclass may predispose to infectious disease. For example, IgG2 deficiency has been related to Haemophilus ~rt,f~~a~~.~t~e type b and pneumococcal infections&dquo;. IgG3 deficiency, in contrast, has been related to a history of recurrent upper and lower respiratory tract infection and bronchial asthma 42. Our results indicate that patients with ARD often have high serum levels of IgG 1, IgG3 and IgG4 and that IgG and IgG3 levels are often elevated in parallel. Raised levels were most often present in patients with SLE, 2° SS and 1 ~~‘, in contrast to patients with RA, myositis and scleroderma. Raised levels of IgG4 were found in only two patients with lupus, but were also high in two other patients with myositis, perhaps suggesting that they had been exposed to chronic antigenic stimulation with protein antigens 43. Some ARD patients had a low level of IgG2. This is in agreement with a previous report of these conditions 44. It was notable that nearly 50% of the patients Thus, ~~C.~l and IgG3
are
isotypes. The pattern of antibodies to Sm/RNP was more ’promiscuous’. was the most common subclass to Sm/RNP in 2° SS and scl~ruder~na. In contrast, raised IgG2 levels were recorded only in I ° SS (I(1~7o) and SLE (8.3%), whereas IgG3 and/or IgG4 against Sm/RNP were present in all of the diseases (IgG4 in 30% of the RA patients). We did not find a correlation between the concentration of IgG subclasses in the sera and recognition of ‘s~elf-~c~~np~n~nts’. Perhaps the high level of IgG in ARD patients with hypergammaglobulinaemia is due to non-specific activation of B cells stimulating multiple clones not all of which are directed against ’selfcomponents’. The high levels of IgGl and IgG3 may be due to the related close genetic linkage of the ’Y1 and y3 genes, implying that stimulation caused a sequential switching among the genes coding for the constant region of the heavy chain within the B-cell genome. Alternatively, the pattern of the Ig~&dquo;r subclasses against ’self-component’ could be due to a polymorphic antigen (such as a super antigen) that triggered this response and the persistence of this antigen could explain the
with both 1 and 20 SS had reduced levels. In contrast, only 10-15% of the patients with SLE, myositis and RA had evidence of IgG2 deficiency. In the SLE patients the levels of IgG subclasses have been reported to show a correlation with susceptibility to infectious disease, notably low levels of IgG3 and IgG4, which are linked to mycotic infection. Oxelius et al.45 found that IgG2 and IgG4 deficiencies were associated with IgA deficiency. Other authors, in contrast, have reported a correlation between disease activity (in SLE) and an increase of IgG antibodies to tetanus and cytomegalovirus&dquo;. Our results show that ARD patients, especially those with SS SLE, have a selective IgG2 deficiency and thus they may be more susceptible to infectious disease than the normal person~. We stratified the SLE and 2° SS patients according to their corticosteroid and azathioprine requirements. Because our sample size is relatively small we are cautious about interpreting the data, but the patients on more than 10 mg of steroid plus azathioprine had low levels of I~~l and IgG3 immunoglobulin compared with those on less than 10 mg or those without any treatment. Large numbers of patients will need to be studied to consider the possible effects of the treatment and disease activity.
high level of IgG4. These results may be compared to the relatively few studies of IgG subclass distribution in patients with infectious disease. For example, Zouali et al.’ showed that in patients with acute malaria 18070 had antibodies to dsDNA and that these wore of the IgGl subclass, while the antibodies to ssDNA (22%) and RNP (32Vo) were more evenly spread the subclasses, especially in the former case. In contrast, IgG2 was found to be the predominant subclass among antibodies binding a .~~yct~~c~ct~ri~~ leprae sonicate extract in the sera of patients with leprosy, although lgd~rl and IgG4 were more prominent in certain disease
subtypes 48
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Acknowledgements This work was supported in part by Programa Universitario de Investi~aeic~n en Salud (PUIS-DGAPA) from the Autonomous National University of M6xico (UNAM), and by the Arthritis and Rheumatism
Council, UK.
antibodies in systemic lupus erythematosus. J Autoimmunity 1988; 1: 483-94. 20. Mater L, Wilhelm JA, Angehrn W, Skvaril F, Schopfer K. Selective antibody deficiency and recurrent pneumococcal bacteriemia in patients with Sjögren’s syndrome, hyperimmunoglobulinaemia G and deficiencies of IgG2 and IgG4. New Engl J Med 1985; 312: 1039-41. 21. Yount WJ, Dyer K, Fuller CR. Distribution of double stranded DNA (dsDNA) antibodies in systemic lupus erythematosus (SLE): restriction to IgM, IgG1 and IgG3. Arthritis Rheum 1983; 26: S73
(abstract D29).
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(Received 9 August 1992) 17 September 1992)
(Accepted
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