Antibodies to CD4 in primary Sjögren's syndrome

G. Henriksson3, R. Manthorpe1 and A. Bredberg

Department of Medical Microbiology, University Hospital, Malmö and
1 Sjögren's Syndrome Research Centre, Department of Rheumatology, University Hospital, Malmö, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. The purpose of this study was to examine whether antibodies against CD4 are present in patients with primary Sjögren's syndrome, and to explore the possible correlation between these antibodies and the CD4+ T lymphocyte depletion that is seen in some Sjögren patients.

Methods. Sera from 214 patients with primary Sjögren's syndrome, 154 healthy blood donors, 38 age- and sex-matched controls without autoimmune disease, and 77 HIV-1-seropositive individuals were analysed by an enzyme-linked immunosorbent assay (ELISA) using recombinant soluble CD4 as the antigen.

Results. Anti-CD4 antibodies were observed more frequently in patients with Sjögren's syndrome (12.6%) as compared with the control groups (0.6%) (P < 0.001), and at a level similar to that seen among the HIV-1 patients (13.0%). However, no correlation was found between the presence of anti-CD4 antibodies and CD4+ T lymphocytopenia in the Sjögren patients.

Conclusion. This is the first study that shows anti-CD4 antibodies in patients with primary Sjögren's syndrome. The significance of these antibodies in the immunopathogenesis of Sjögren's syndrome remains to be determined.

KEY WORDS: Anti-CD4 antibodies, Autoimmunity/autoantibodies, Sjögren's syndrome.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The cell surface CD4 glycoprotein defines a major functional subset of mature T lymphocytes known as helper T cells, and is thought to interact with class II major histocompatibility complex (MHC) molecules mediating efficient association between helper T cells and antigen-presenting cells. CD4 is an integral membrane protein sharing significant amino acid and structural homologies with members of the immunoglobulin supergene family [1].

In human immunodeficiency virus (HIV) infection, the progressive loss of peripheral CD4+ T lymphocytes is a characteristic finding [2]. However, several reports have presented patients with circulating CD4+ T lymphocytopenia in the absence of serological evidence of HIV infection, defined immunodeficiency, or therapy associated with T cell depletion [3, 4], a condition termed idiopathic CD4+ T lymphocytopenia.

Primary Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by diminished function of exocrine glands often associated with focal lymphocytic infiltration. However, many organs and tissues outside these primary gland target sites may additionally be affected, leading to symptoms and signs from both exocrine and non-exocrine organs 5, 6].

In a recent study we reported persistent decreased levels of CD4+ T lymphocytes in peripheral blood in six of 115 (5.2%) patients with primary SS without serological evidence of HIV infection [7]. Even if this prevalence of CD4+ T lymphocytopenia is fairly low, it is significantly higher than the rate reported for any other non-HIV patient or population group previously investigated.

It has been suggested that cytotoxic anti-lymphocyte antibodies restricted to the CD4+ subpopulation of T lymphocytes are involved in the pathogenesis of idiopathic CD4+ T lymphocytopenia [8, 9]. A great number of HIV-infected patients display antibodies against CD4+ lymphocytes [1016] as well as autoantibodies typical of non-organ-specific autoimmune diseases [17]. The relevance in HIV infection of these anti-CD4 cell antibodies is unknown, but an association with CD4 cell depletion has been reported [16]. In addition, there has been sporadic reports of patients with systemic lupus erythematosus (SLE) having anti-lymphocytic antibodies that preferentially react with CD4+ lymphocytes [11, 18].

Accordingly, the question was raised whether SS patients also display antibodies against CD4+ lymphocytes. The aim of the present study was to examine the presence of antibodies against recombinant soluble CD4 in patients with primary SS using an enzyme-linked immunosorbent assay (ELISA), and to explore the possible correlation of these antibodies with CD4+ T lymphocyte depletion.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and controls
Frozen sera from 214 patients with primary SS were analysed for anti-CD4 antibodies using an ELISA. There were 185 females and 29 males, aged 14–86 yr (mean 59 yr). The diagnosis of primary SS was made according to the Copenhagen criteria, i.e. at least two abnormal objective tests of lacrimal gland function and two abnormal tests of salivary gland function [19]. Five of the six (one deceased) primary SS patients previously found to have CD4+ T lymphocytopenia, with a CD4 count of less than 300 cells/µl or less than 20% of the total lymphocyte count at more than one determination [7], were included among the 214 patients studied. Two panels of controls were used. The first was composed of 154 randomly selected sera from Malmö University Hospital's blood bank. The second was composed of sera from 38 persons without autoimmune disease, which were age and sex matched with the SS patients [mean age 61 (range 39–86) yr, 34 females/4 males]. Serving as a positive control for the ELISA used in this study, sera from 77 HIV-1-seropositive individuals were also examined. The presence of anti-HIV-1 antibodies was assessed and confirmed by different commercial ELISAs and Western blots.

Recombinant soluble CD4
Recombinant soluble CD4 was a gift from R. W. Sweet (SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA). This molecule contains the extracellular portion consisting of an amino-terminal variable (V)-like domain, a joining (J)-like region and a third extracellular domain but lacks the hydrophobic transmembrane domain homologous to class II MHC beta chains and the highly charged cytoplasmic segment of native CD4. The CD4 preparation was >95% pure, as estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis [20].

Detection of anti-CD4 antibodies by ELISA
Polystyrene microwell plates (F96 Maxisorp, Nunc-Immuno Module, Roskilde, Denmark) were coated overnight at 4°C with 50 ng per well of recombinant soluble CD4 dissolved in 100 µl of 0.1 M NaHCO3 buffer (pH 9.4). The plates were then washed four times with washing buffer [phosphate-buffered saline (PBS) containing 0.05% Tween 20, pH 7.4] and incubated for 1 h at room temperature with 200 µl of blocking buffer (PBS containing 1.5% ovalbumin and 0.05% Tween 20, pH 7.2). After renewed washing, 100 µl per well of patient sera diluted 1:500 in blocking buffer was added. A positive control, mouse anti-CD4 monoclonal antibodies (anti-human Leu3a, Becton-Dickinson, San Jose, CA, USA) diluted in blocking buffer (three different dilutions) was included on each plate. One of these three dilutions of the positive control was used as a calibrator in order to reduce the inter-assay variation. Thus, when the observed optical density (OD) value of the calibrator deviated from its assigned OD value, the OD values of patient samples belonging to the same ELISA plate were corrected for accordingly. A negative control, anti-CD8 monoclonal antibodies (anti-human Leu2a, Becton-Dickinson) was also run with each plate. The sera and controls were allowed to react at room temperature for 1 h. The plates were then washed four times with washing buffer and incubated once more for 1 h at room temperature with 100 µl per well of peroxidase-conjugated rabbit anti-human IgG or peroxidase-conjugated rabbit anti-mouse IgG (DAKO A/S, Glostrup, Denmark) diluted 1:6000 in blocking buffer. Finally, the plates were washed again and 100 µl of 20 mM tetramethylbenzidine in citrate buffer (0.1 M, pH 4.25) with 2 mM H2O2 at a 1:20 ratio was added as the substrate. The enzymatic reaction was stopped 5 min later by the addition of 100 µl of 1 M H2SO4 and the OD at 450 nm was determined by a spectrophotometer (Multiskan Plus, Labsystems, Finland).

All sera and controls were analysed in triplicate, of which two were run in wells coated with CD4 and one in an uncoated well (the background) on the same plate. The result was expressed as the OD difference between the two coated wells (mean value) and the uncoated well. In this way, any false-positive reactions due to non-specific binding of patient IgG to factors employed in the assay other than CD4 were excluded for. The control groups constantly had a low background, while a slightly higher variation in background activity was noted in the SS group. Due to the variability of the method [for coefficient of variation (CV) see below], for some patients lacking anti-CD4 IgG a negative result will therefore be obtained. The data presented in Fig. 1Go include these negative figures, in order for the variability of the method to be visualized.



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Fig. 1. Autoantibodies against recombinant soluble CD4 determined by ELISA. Individual patient OD values are shown. The white squares represent primary SS patients with a low number of peripheral blood CD4-positive lymphocytes, and the black circles those with a normal CD4 cell count.

 
Checkerboard titration of antigen concentration vs serum dilution showed 50 ng CD4 well coating and 1:500 dilution of patient sera to be optimal (data not shown).

The inter-assay CV (S.D./mean OD) was estimated using one of the three dilutions of the positive control. Thirty-eight determinations, made on 38 separate plates and on 29 separate dates, showed a CV of 12.2%.

Flow cytometry and other immunological tests
Peripheral blood lymphocytes were analysed using an Epics-Profile II flow cytometer (Coulter Corp., Hialeah, FL, USA) with dual colour (fluorescein isothiocyanate and phycoerythrin) direct immunofluorescence and mouse monoclonal antibodies (Dakopatts A/S or Coulter Corp.) as previously described [7]. CD4+ T lymphocytopenia was defined as an absolute CD4+ T cell level <300 cells/µl or <20% of the total lymphocyte count at more than one determination.

Analysis of antinuclear antibodies (ANA) was performed by indirect immunofluorescence with a fixed preparation of HEp-2 cells as the substrate, purchased from Immunoconcept (Sacramento, CA, USA).

The presence of antibodies against extractable nuclear antigen (ENA) was determined by immunodiffusion and/or ELISA, also using reagents obtained from Immunoconcept.

Rheumatoid factor (RF) was analysed by the Waaler-Rose test (an in-house method).

The performance of these analyses was controlled owing to our participation in a national quality assessment scheme and the international UK scheme (U.K. NEQAS).

Statistical analysis
The Pearson {chi}2-test and the Mann–Whitney test were used for statistical analysis of the data [21]. A difference was considered statistically significant if P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ELISA analysis of antibodies against recombinant soluble CD4
In the control group comprising 154 randomly selected sera from healthy blood donors, the concentration of anti-CD4 antibodies was found to be very low (the ELISA OD mean value was 0.006) and to fit a normal distribution (Fig. 1). In order to be able to identify individuals with high anti-CD4 antibody levels, a positive reaction was identified by a value exceeding 3.29 S.D. within this control population, corresponding to a 99.9% confidence interval. Using this definition, a threshold OD value of 0.045 was obtained. Based on this threshold, antibodies to CD4 were detected in one (0.6%) of these 154 control sera (Fig. 1). Even in the control group composed of 38 age- and sex-matched sera, the ELISA OD mean value was very low (0.003) and not a single one was positive for anti-CD4 antibodies (data not shown).

The share of positive patients in the primary SS group (27/214, 12.6%) (Fig. 1) was higher compared with the control group of healthy blood donors (1/154, 0.6%). This difference was statistically significant (P < 0.001, Pearson {chi}2-test). This higher proportion of positive sera yielded a higher mean OD for the SS group (0.020) than found for the control group (0.006). The statistical significance of this difference was, however, not possible to test by t-test, since the distribution of values was highly skewed. A difference in location was supported by the Mann–Whitney test, which indicated significant differences in medians (P < 0.01). Of the 27 SS patients who were positive for anti-CD4 antibodies, there were three males (11.1%). No correlation was observed between the anti-CD4 antibody level and patient age in the SS group. Linear regression analysis of these two parameters yielded a coefficient of correlation of 0.12 (data not shown).

In the group composed of 77 HIV-seropositive patients, 10 (13.0%) were considered anti-CD4 positive (Fig. 1). This share of positive patients was also higher compared with the control group and the difference was statistically significant (P < 0.001).

No statistically significant difference between the share of positive patients in the primary SS group and the HIV group was found.

None of the five primary SS patients reported by Kirtava et al. [7] to have CD4+ T lymphocytopenia displayed antibodies to recombinant soluble CD4. Among the total of 214 primary SS patients, additionally three patients with CD4+ T lymphocytopenia were found. None of these three patients had anti-CD4 antibodies detectable by ELISA. Thus, no correlation between the occurrence of anti-CD4 antibodies and CD4+ T lymphocyte depletion in patients with primary SS was apparent (the white squares in Fig. 1 represent those with CD4+ T lymphocytopenia among the primary SS patients).

ANA, ENA, RF, serum Ig, and flow cytometric analyses
Analyses of ANA as well as ENA were performed in 199 primary SS patients, of whom 26 were positive and 173 negative for anti-CD4 as determined by ELISA (Table 1Go). Among these 199 patients, RF determination and flow cytometric analysis of peripheral blood lymphocytes were also performed on 184 and 172 patients, respectively.


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TABLE 1. Relation of ANA, ENA and RF findings to anti-CD4 in the primary SS patients

 
The mean percentage of CD4+ T cells (of total lymphocytes) was 41.2 in the anti-CD4-positive group, and 44.3 in the anti-CD4-negative group (data not shown). The mean absolute level of circulating CD4+ T cells was 0.79 x 109 cells/l in the anti-CD4-positive group, and 0.89 x 109 cells/l in the anti-CD4-negative group (data not shown). These differences between the anti-CD4-positive and anti-CD4-negative groups were not statistically significant. Furthermore, linear regression analysis of the anti-CD4 antibody level with the number of CD4+ T lymphocytes displayed no correlation (Fig. 2Go). Consequently, primary SS patients positive for anti-CD4 antibodies were not found to have a lower number of circulating CD4+ T lymphocytes than anti-CD4-negative primary SS patients.



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Fig. 2. No correlation between the level of anti-CD4 antibodies determined by ELISA and the number of circulating CD4+ T lymphocytes, as shown by linear regression analysis (r = - 0.08) in 172 patients with primary SS.

 
Neither did a statistically significant difference in the frequency of patients with ANA or ENA (only anti-Ro/SS-A and anti-La/SS-B were observed) or with different types of ANA pattern (i.e. speckled, homogeneous, etc.) appear between the anti-CD4-positive and anti-CD4-negative groups (Table 1). It was apparent, however, that 11 of the 12 anti-CD4-positive primary SS patients with speckled ANA pattern also had anti-Ro/SS-A and/or anti-La/SS-B. This was found to a lesser degree (in 52 of 64 patients) in the anti-CD4-negative primary SS group.

It is well known that SS patients often have hyperglobulinaemia, but no correlation was apparent between the levels of anti-CD4 antibodies and serum IgG, IgA, and IgM (coefficient of correlation 0.12, 0.16 and 0.22, respectively) (data not shown). In the anti-CD4-positive group comprising 27 primary SS patients, the mean levels of serum IgG, IgA and IgM were 15.1, 3.1 and 1.3 g/l, respectively, while the mean levels were 12.6, 2.4 and 1.0 g/l in the anti-CD4-negative group (data on serum Ig levels were available in 168 primary SS patients negative for anti-CD4 antibodies). These differences between the two groups were not statistically significant (data not shown).

Finally, no correlation between anti-CD4 ELISA positivity and RF reaction was found (Table 1).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using an ELISA technique with highly purified recombinant soluble CD4 as the antigen we detected serum IgG antibodies against CD4 in 12.6% of the patients with primary SS. As a positive control of the ELISA test used in this study, sera from 77 HIV-seropositive individuals were examined; 13.0% of these individuals also displayed anti-CD4 antibodies. Corresponding results among the majority of previous reports have been 5.2–20% [1014], although some investigators have found an even higher proportion of anti-CD4 antibodies in HIV-infected patients [15, 16].

It may be argued that the relatively low ELISA OD levels observed by us constitute a weakness of the presented evidence, making any conclusions drawn unjustified. However, several facts speak against this argument:

(i) the accuracy of our ELISA method is high enough (CV 12.2%) for the primary SS OD values to be significantly different from those of the control group;
(ii) the subtracted ELISA background reactivity did include not only that seen using buffer instead of serum, but also (for all patients and controls) the non-specific binding of serum to an uncoated (i.e. without CD4) microplate well. This procedure was adopted in order to reduce as much as possible signals unrelated to IgG–CD4 binding, and this will reduce the level of the recorded OD values;
(iii) similar OD levels were seen in the HIV and primary SS patient groups. This applies to both the group mean values and the individual values presented by ELISA-positive patients.

Summarizing these methodological considerations, our results are in accordance with previous reports and consequently we regard our ELISA technique as an accurate method.

To the best of our knowledge, this is the first study that shows measurable levels of anti-CD4 antibodies in the sera from patients with primary SS. Sporadic SLE cases presenting with anti-lymphocytic antibodies that preferentially react with CD4+ lymphocytes have previously been reported [11, 18]. For one of these patients, there was also a relative depletion of CD4+ cells [18]. In the group of primary SS patients studied by us, there was no evidence of an effect on CD4+ cells by anti-CD4 antibodies, since there was no sign of a correlation between anti-CD4 antibodies and CD4+ T lymphocytopenia. There have also been observations in two SLE cases [22], as well as in two HIV patients [23], of hereditary forms of CD4 epitope deficiency, causing low flow cytometry CD4 cell counts.

A CD4+ lymphocyte deficiency is not the only sign known to be shared by primary SS and HIV patients. There have also been reports of SS-like symptoms and ANA positivity in HIV patients [24], and indications in SS of herpesvirus and retrovirus components [25]. Certain individuals respond to HIV-1 infection by developing circulating and tissue infiltrative CD8 T cell lymphocytosis. This disorder, termed diffuse infiltrative lymphocytosis syndrome (DILS), may present as a sicca syndrome in HIV-infected individuals [26]. The present findings of anti-CD4 antibodies in SS is another feature common to HIV and SS patients, lending further support for the role of a virus in this disorder.

The mechanism of production of autoantibodies is not fully known. T cell tolerance to self-antigens can be reached by elimination of self-reactive T lymphocytes in the thymus (clonal deletion) or through peripheral T cell anergy [27, 28]. Both mechanisms require that self-antigen is processed to become bound with high affinity to MHC molecules. According to present knowledge of antigen processing, a large number of self-determinants are cryptic in the sense that they are not adequately exposed by cell surface MHC, permitting T cells specific for these cryptic epitopes to escape from clonal deletion. Therefore, altered cellular processing of self-antigens may lead to activation of autoimmune T cells [29, 30]. Salemi et al. [31] have shown that this mechanism may apply for CD4. In this study, T helper cells were exposed to HIV gp 120 antigen, causing down-regulation of plasma membrane CD4 and alteration of CD4 processing, thus making cryptic epitopes visible to autoreactive T cells.

It is tempting to apply this concept of cryptic peptide autoimmunity to our finding of anti-CD4 antibodies in primary SS patients. Maybe some mechanism that changes the processing of the CD4 molecule as an antigen and/or increases the yield of previously ‘invisible’ CD4 peptides is capable of inducing an autoimmune-specific T cell response.

Altered expression of the La/SS-B gene in an SS patient, by way of intron-located promoter switching leading to modification of mRNA processing, has been reported [32]. This finding, viewed together with our knowledge of spliceosome and related RNA-processing nucleoproteins as frequent targets of the ANA observed in SS (and other systemic connective tissue disorders) [33], suggests another possible mechanism (i.e. in addition to the cryptic peptide observations) for cell surface MHC expression of altered self-antigen, generating autoimmune T cells.

In summary, our observation of anti-CD4 autoantibodies in patients with primary SS is of interest considering that CD4 is an essential regulatory molecule of the immune system and consequently it is possible that such autoantibodies contribute to lymphocyte dysfunction in SS. However, the significance of these autoantibodies in the immunopathogenesis of SS remains to be determined.


    Acknowledgments
 
This work was supported by the Swedish Cancer Society, Malmö General Hospital Cancer Foundation, the Crafoord Foundation, and ‘Förenade Liv’ Mutual Group Life Insurance Company.

We thank Dr Ray Sweet (SmithKline Beecham Pharmaceuticals) for the generous gift of recombinant soluble CD4 and Dr Leo Flamholc (Department of Infectious Diseases, Malmö) for the gift of sera. We are grateful to Marta Brant for excellent technical assistance, Karin Brundell-Freij for expert statistical assistance and Henrique Santos for kind assistance with patient serum Ig data.


    Notes
 
3 Correspondence to: G. Henriksson, Department of Medical Microbiology, University Hospital, S-205 02 Malmö, Sweden. Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Submitted 18 January 1999; revised version accepted 8 September 1999.