An increased prevalence of autoimmune diseases (AD) such as rheumatoid arthritis [1] (RA) or systemic sclerosis [2] has been demonstrated in silica-exposed patients from the beginning of the century. Anti neutrophil cytoplasmic antibodies (ANCA) associated diseases have been reported [3] and silica dust exposure was reported by 46% of patients with ANCA-associated small vessel vasculitis compared with 20% of control subjects [4]. We reported on ANCA-associated glomerulonephritis in former slate workers [5].

The pathophysiology of pulmonary silicosis is wellknown. Following inhalation, silica particles enter the lung and are subsequently found in the alveolar space, the lung tissue and the pulmonary lymphatics. Alveolar and interstitial macrophages are activated after particle uptake; they produce reactive oxygen species, lysosomal agents, pro-inflammatory or pro-fibrotic cytokines such as IL-1, TNF-α, TGF‐β, PDGF and products of arachidonic acid metabolism [6]. The recycling of the silica particles by other macrophages, the recruitment of various inflammatory cells, the development of pulmonary fibrosis and of granulomas with profound alteration of lung structure are the following steps leading to the pulmonary disease. Moreover silica particles can be shown in tiny quantities in the blood stream [7], and extrapulmonary lesions have been described with silica nodules present in liver, spleen, extra thoracic lymph nodes and bone marrow [8]. In contrast with the function of macrophages in the course of the disease, little is known about the role of lymphocytes in silicosis or about the mechanisms leading to the development of autoimmune disorders. Macrophages, B and T cell subsets have been described in the lung and bronchoalveolar lavage fluid after acute exposure [9] but very few data are available in blood [10].

So we have studied a cohort of assessed silicotic men in steady state in order to determine the prevalence of clinical and immunological disorders and to look for differences in lymphocyte subsets in blood when compared with non-exposed controls.

The control group (group 2) was composed of volunteer men without any history of professional silica exposure (clergymen, third age university students, retired military and retired teachers). The same mailing procedure was used to get in touch with them, and their agreement was obtained in the same way. Men over 77 years or presenting acute evoluting diseases were excluded from the study. Our hospital ethic committee gave consent to the study.

Silicosis was assessed according to the International Labor Office criteria: combination of long-lasting (at least 5 years) exposure to silica in slate mining with characteristic abnormalities on chest X-ray. A three physician college assessed the percentage of the impairment related to silicosis.

Medical examinations with specific search for overt AD were carried out by the same physician (JFS) for all patients in the morning, and afterwards urine and blood samples were collected. The prevalence of smoking, of urogenital and cardiovascular diseases was identical in both groups. In the same way, the prevalence of medications other than immunosuppressives was similar in the two groups. The mean age of the control group (67 years ± 4·5) was significantly lower when compared to that of the silicotic group (70 years ± 3·5). Body mass index was within the normal range and did not differ between the two groups.

Rheumatoid Factors (RF), C3 and C4 complement components were measured in serum by nephelometry (Dade Behring) and serum protein immunoelectrophoresis was achieved.

The plasmatic interleukin-1 beta (IL-1β) was measured by the ELISA TiterZyme IL-1β EIA Kit (Bio Advance Emerainville France). Circulating angiotensin converting enzyme (ACE) was quantified by spectrophotometry (Sigma diagnostic), normal values are: 30 ± 11 U/l. Antinuclear antibodies (ANA) were detected by indirect immunofluorescence (IIF) on HEp2 cells, starting the screening at a dilution of 1/100, and antidouble strand anti-DNA antibodies were detected by IIF on Crithidia luciliae. ANCA were detected by IIF on ethanol fixed normal human neutrophils, antimyeloperoxidase (MPO) and antiproteinase 3 (PR-3) antibodies by ELISA (Euroimmun Gross Groenau, Germany).

Quality control for immunophenotyping was performed by verifying internal consistency for CD3⁺ lymphocytes present in each reaction tube containing the CD3 monoclonal antibody. The sum of CD3⁺, CD19⁺ and CD16/CD56⁺ percentages was approximately 100 ± 5% for each sample analysed.

The prevalence of AD history was significantly higher in group 1 as compared to group 2: 11% versus 0% (P < 0·05). As shown in Table 1, six patients exhibited some form of AD; 52 subjects were AD free. Three patients (patient nos 2, 3 and 4) had an active seropositive RA under treatment, one (patient no. 1) suffered a documented sensory neuropathy of unknown origin and RA in remission at the time of the study. The fifth patient had biopsy proven pauci immune glomerulonephritis. The sixth patient, who had myasthenia gravis with circulating antilaminin, antismooth and antistriated muscle antibodies but no antiacetylcholine antibodies, had manifested clinical improvement under prolonged prostigmin therapy. These two latter patients were treated with steroids and the three patients suffering from active RA were treated with gold salts plus steroids (patients no. 3 and no. 4) and anti‐malarial drug (patient no. 2). All six patients had high ANA titre, but no ANCA were detected.

Table 1 Patients with medical history of autoimmune disease

Table 2 Biological data

Table 3 Blood cell counts and cell subsets percentage

These clinical data are in agreement with those previously reported by others although none of our patients had either systemic sclerosis, lupus syndrome or systemic vasculitis, but the prevalence of these AD in the silicotic population is very low (about 0·2%) [13]. The biological and immunological abnormalities observed in our population have also been previously described [11,12 14–17, ]. In a retrospective study (unpublished data) we found a 13% (11/85) prevalence of professional silica dust exposure in men over 40 years admitted in our ward for primary glomerulopathy compared to a 2·3% prevalence in the general population of the district (P = 4·3 × 10^−6, χ² test). Circulating ANCA with antimyeloperoxidase specificity were detected in five of 10 of these patients. These data are in agreement with the results of Hogan et al. in a study conducted in the south-east of United States [4].

The major finding that emerge from this study is the very significant decrease in the number of the different lymphocyte subsets and the increased ratio of activated T cells in the blood. To the best of our knowledge this has not been reported before. Only Watanabe et al. (1987) [10] reported on the occurrence of a significant decrease in peripheral OKT3⁺ cells count. Struhar et al. (1989) [9] and Sanchez-Roman et al. (1993) [18] did not report on lymphopenia in their studies.

These results raise two important questions concerning the mechanism of lymphopenia and its potential role in the occurrence of AD. Malnutrition is an unlikely hypothesis for lymphopenia since the body mass index of the two groups was equivalent; the difference in the mean age between the two groups (3 years) is too weak to explain the difference observed [19].

The increased percentage of activated T cells could reflect a chronic stimulation of T cells by silica particles liberated from damaged macrophages [8]. Interestingly it has been shown [20] that silica may act as a superantigen and that lymphocytes incubated with silicate became apoptotic through the Fas/Fas ligand pathway [21]. Moreover serum levels of soluble Fas ligand are elevated [22] and an overexpression of soluble Fas mRNA [23] have been observed in peripheral blood mononuclear cells of silicotic individuals. Apoptosis could very well explain the T cell lymphopenia observed in this study; however, it does not explain the decrease in B and NK cell counts. Lymphocyte sequestration in enlarged silicotic nodules [8] could be another potential mechanism of lymphopenia.

The other point to be discussed concerns the potential role of lymphopenia in the occurrence of autoimmunity. Several experimental data now strongly support the role of lymphopenia in autoimmune diseases. Lymphopenia, whether congenital in BB rats or experimentally induced by irradiation, thymectomy or cyclophosphamide, is commonly associated with the onset of autoimmune disorders (review in [24]). Furthermore, in some experimental models, the disease can be prevented by reconstitution with cells of a specific phenotype [25]. The importance of the CD4⁺ CD25⁺ subset in the maintenance of immunological self tolerance is crucial [26] since elimination of CD25⁺ T cells in normal mice leads to the development of autoimmune diseases. It is interesting that in our population the absolute number of CD25⁺ cells was diminished to the same extent as the other lymphocyte subsets.

In coeliac disease, Di Sabatino [27] showed a decrease in the absolute number of CD3⁺, CD4⁺, CD8⁺ and CD19⁺ cells in untreated patients as compared with treated patients and healthy volunteers. The proportion of CD3⁺ HLA DR⁺ cells was found higher in untreated patients than in controls (12·8% ± 7 versus 6·2% ± 3). Furthermore, in systemic lupus erythematosus and primary vasculitides, a decrease in total lymphocyte count related to activation induced cell death has been demonstrated in vitro [28]. These data are reminiscent of our own.

It is noteworthy that lymphopenia was observed in 48/53 of silicotic subjects while only 10% develop overt clinical autoimmune disorders. This indicates that other genetic (MHC?) or environmental factors are required for the development of autoimmune diseases.

In conclusion, our results show that in men with occupational pulmonary silicosis the absolute number of circulating T, B and NK cells was dramatically reduced when compared to healthy volunteers. The percentage of each lymphocyte subset was identical in the two groups except for the percentage of activated T cells that was significantly increased in the silicotic group. The presence of lymphopenia and relative increase in circulating activated T cells could be part of different factors that initiate the onset of autoimmune disorders in this population.

The authors thank Pr. P. Druet and Dr A. Saoudi for their comments and suggestions on this manuscript. This work was supported by ‘Programme Hospitalier de Recherche Clinique 1994’.