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Clin Exp Immunol. Oct 1998; 114(1): 113–118.
PMCID: PMC1905085

Costimulatory molecules in Wegener's granulomatosis (WG): lack of expression of CD28 and preferential up-regulation of its ligands B7-1 (CD80) and B7-2 (CD86) on T cells

Abstract

T cells are most likely to play an important role in the pathogenesis of WG, and recently a predominant Th1 pattern of immune response has been demonstrated in granulomatous inflammation. Since the expression of costimulatory molecules has a significant impact on the cytokine profile and proliferation response of T cells, the goal of this study was to characterize the expression of costimulatory molecules (CD28, CTLA-4 (CD152), B7-1 (CD80), B7-2 (CD86)) on T cells, monocytes and B cells in WG, and to correlate the findings with clinical parameters such as disease activity, extent and therapy. WG patients (n = 24) and healthy controls (HC; n = 17) were examined for the expression of costimulatory molecules by fluorescence-activated cell sorter analysis, both in whole peripheral blood and after in vitro activation of T cells and antigen-presenting cells. Results were correlated with clinical data. The expression of CD28 on CD4+ and CD8+ cells was significantly lower in WG than in HC (CD28+ 81.4% in WG versus 97.9% of CD4+ cells (P < 0.0001); CD28+ 44.6% in WG versus 68.5% of CD8+ cells (P < 0.00001)), both in peripheral blood and after in vitro activation. A lower percentage of monocytes was B7-2+ in WG than in HC in peripheral blood, whereas no significant differences in the expression of B7-1 and B7-2 were observed after in vitro stimulation of monocytes and B cells. After in vitro activation a significantly higher percentage of B7-1+ and B7-2+ T cells was seen in WG. There was no significant difference in the CTLA-4 expression pattern between WG and HC. The percentage of CD28+ lymphocytes correlated negatively with the Disease Extent Index cumulated over the course of disease (r = −0.46, P = 0.03), indicating a more severe manifestation in patients with lower CD28 expression. Correlations with other clinical parameters such as activity or therapy were not seen. WG patients show a lack of CD28 expression on T cells and an unusual up-regulation of its ligands B7-1 and B7-2 on T cells after in vitro activation as well as a lower expression of B7-2 on freshly isolated monocytes compared with HC. These features might promote the Th1 cytokine pattern and thereby contribute to persistently high levels of immune activation in WG.

Keywords: Wegener's granulomatosis, costimulatory molecules, CD28, B7

INTRODUCTION

A large body of evidence indicates that T cells play a critical role in inflammatory processes in WG. They are abundant in inflammatory lesions and the Th1 pattern predominates in granuloma-derived T cell clones [1]. Furthermore, a high percentage of peripheral blood T cells is activated, as can be seen by their expression of HLA-DR and CD25 (IL-2 receptor), both markers for T cell activation [2]. Even the cleavage product of CD25 (sIL-2 receptor) is elevated in active WG, as is the soluble CD30 molecule, which is another marker of T cell activation. The plasma levels of both markers correlate closely with disease activity [3,4].

Activation of T lymphocytes involving both cytokine production and proliferation requires two signals: one through the T cell receptor–CD3 complex and at least one costimulatory signal involving the B-7 (CD80, CD86) family of molecules on antigen-presenting cells (APC) and the CD28 and CTLA-4 (CD152) receptors on mature T cells. Recently the role of abnormal expression of costimulatory molecules in chronic immunological disorders was described. CD4+CD28 T cells were found to be more frequent in rheumatoid arthritis (RA) patients than in healthy controls (HC), while the expansion of these cells correlated with extra-articular disease involvement [5,6]. In systemic lupus erythematosus (SLE), an interferon-gamma (IFN-γ)-induced reduction in the expression of B7-1 (CD80) on APC compared with controls has been demonstrated which might contribute to defective APC function in vitro [7]. On the other hand, blockade of B7 ligands using MoAbs has been shown to prevent the development and progression of lupus in lupus-prone mice [8].

The aim of the present study was to characterize the expression of CD28, CTLA-4 and their ligands B7-1 and B7-2 on T cells, monocytes and B cells in patients with WG.

PATIENTS AND METHODS

Patients and controls

We examined 24 patients with histologically proven c-ANCA (proteinase 3 (PR3)–anti-neutrophil cytoplasmic antibody (ANCA))-positive generalized WG from our department and 17 healthy controls. All WG patients fulfilled the criteria of the American College of Rheumatology [9] and of the 1992 Chapel Hill Consensus Conference definition [10]. Seven of the WG patients were women and 17 men (HC, five women, 12 men). Their mean age was 56 years (range 36–75 years; mean age of HC 57 years, range 34–75 years). The mean duration of disease was 5.1 years. Nine of the patients were in active state and 15 in inactive state of disease. Disease activity was determined using standard laboratory parameters (C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)) as well as clinical findings and imaging procedures. Twenty-two of the patients were treated with prednisolone, 10 received cyclophosphamide and eight methotrexate. Organ involvement was measured using a cumulative Disease Extent Index (DEI [11]). Briefly, all organ manifestations during the course of disease were allocated two points, constitutional symptoms received one point. The maximum possible cumulative DEI score was 21 points. For this purpose the histories of the 24 patients were reviewed.

Cell isolation and culture

Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll gradient centrifugation. The PBMC were cultured on 24-well tissue culture plates at 37°C for 3 days in RPMI 1640 supplemented with 10% fetal calf serum (FCS) at a concentration of 1 × 106 cells/ml. To stimulate the cells, phytohaemagglutinin (PHA, 1 μg/ml; Sigma Chemical Co., St Louis, MO) and phorbol-12-myristate-13-acetate (PMA, 0.5 ng/ml; Sigma) were added.

For the examination of APC, PBMC were depleted of T cells using immunomagnetic Pan-T beads (CD2, Dynabeads M-450; Dynalab, Oslo, Norway) according to the manufacturer's instructions. Purity of the non-T cell preparation was confirmed by flow cytometry and amounted to < 1% CD3+ cells. Non-T cells were analysed directly after isolation and after 24 and 48 h incubation at 37°C in the presence of 1000 U/ml IFN-γ (Bender, Vienna, Austria) at a concentration of 1 × 106cells/ml.

Flow cytometry

FITC-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD14 and anti-CD19 and PE-conjugated anti-CD28 were obtained from Becton Dickinson (San Jose, CA). FITC-conjugated IgG1 and PE-conjugated IgG2a as well as FITC-conjugated anti-CD4 and PE-conjugated anti-CD3 (all Becton Dickinson) were used as negative and positive controls, respectively. PE-conjugated anti-CD80, anti-CD86, and anti-CD152 were obtained from PharMingen (Hamburg, Germany). Flow cytometry was performed on a EPICS profile II (Coulter, Hialeah, FL).

Gates for positive and negative populations were set in a similar fashion for all groups. Isotype control antibodies were used to determine any non-specific binding.

Peripheral whole blood was incubated for 30 min at 4°C with the appropriate MoAbs. After lysis (Q-Prep; Coulter) the cells were analysed.

The cultured cells were washed and resuspended in PBS at a concentration of 1 × 106cells/ml. After staining for 30 min at 4°C, cells were washed in PBS supplemented with 0.5% bovine serum albumin (BSA) and fixed in PBS containing 1% formaldehyde. Analysis was performed within 24 h.

Statistical analysis

The non-parametric Mann–Whitney test was used to compare the expression of surface markers by WG patients versus controls. Analysis was performed applying a Statistica software package.

RESULTS

CD3+, CD4+ and CD8+ cell frequency in WG and HC

In whole peripheral blood no statistically significant (P = 0.09 CD4+ and P = 0.09 CD8+) difference was observed in the frequency of CD3+, CD4+ and CD8+ cells between WG patients and HC, although the mean CD4+ frequency was lower and the CD8+ frequency higher in WG patients than in HC (Table 1).

Table 1
Percentage of CD3+, CD4+, CD8+ and CD19+ lymphocytes and s.d. in WG and healthy controls (HC) in peripheral blood. None of the observed differences were significant

Lack of CD28 expression on T cells

As shown in Fig. 1, the frequency of CD28+ T cells was significantly lower in WG patients than in HC in CD3+, CD4+ and CD8+ (CD8high, no natural killer (NK)) cells. The frequency of CD4+CD28+ cells was 81.4 ± 11.9% (mean ± s.d.) in WG patients compared with 97.9 ± 1.8% in HC, while that of CD8+CD28+ cells was 44.6 ± 11.8% in WG patients versus 68.5 ± 6% in HC. The frequencies of these cells exhibited a bimodal distribution pattern in both WG patients and HC, although neither group had numbers sufficient to prove this mathematically (data not shown). In addition to the increased frequency of CD4+CD28 and CD8+CD28 cells in WG patients, the mean fluorescence intensity (MFI) of the CD28+ cells as a measure of the antigen density on the cell surface was also lower in WG patients than in HC (1.5 versus 1.9). This difference was not statistically significant (P = 0.08), but a strong correlation was evident between the percentage of CD28+ lymphocytes and the MFI (r = 0.77, P < 0.0001), indicating not only that the WG patients had an increased CD28 T cell population but that even the CD28+ cells expressed lower amounts of this receptor on their surface. This correlation was not seen in HC.

Fig. 1
Box plots of the CD28+ rate of CD3+, CD4+ and CD8+ cells in healthy controls (HC) and WG.

Preferential up-regulation of B7-1 and B7-2 on T cells in WG uponin vitro stimulation

T cells in both groups expressed very low levels of B7-1, B7-2 and CTLA-4 (< 1%). After in vitro activation the expression of CD28 on CD3+, CD4+ and CD8+ cells was of about the same magnitude as it was in whole blood in both HC and WG patients. No up- or down-regulation of this marker was observed (Fig. 2). CTLA-4 was up-regulated upon activation, but no significant difference between the two groups was seen. B7-1 and B7-2 were also up-regulated, the difference for these two molecules being statistically significant: WG patients had a higher frequency of B7-1+ and B7-2+ T cells than HC, with a higher expression on CD8+ than on CD4+ cells (Fig. 2).

Fig. 2
The expression of CD28, CTLA-4 (CD152), CD80 (B7-1) and CD86 (B7-2) on T cells in WG compared with healthy controls (HC) after in vitro stimulation for 3 days, on CD4+ (a) and CD8+ (b) cells.

Freshly isolated monocytes in WG patients expressed significantly lower levels of B7-2

After T cell depletion, non-T cells were analysed after 24 and 48 h. CD19+ cells (B cells) and CD14+ cells (monocytes) expressed only very low levels of B7-1 after isolation. B7-2 expression was low on freshly isolated B cells. B7-2 was expressed on freshly isolated monocytes, but there was a statistically significant lower expression in WG patients than in HC. This difference vanished after in vitro activation of the cells. Upon activation, B7-1 and B7-2 were up-regulated on B cells and monocytes (Fig. 3).

Fig. 3
Expression of CD80 (B7-1, (a)) and CD86 (B7-2, (b)) on freshly isolated B cells (0 h) and after in vitro stimulation (24 h and 48 h). Expression of CD80 (B7-1, (c)) and CD86 (B7-2, (d)) on freshly isolated monocytes (0 h) and after in vitro stimulation ...

Correlation between percentage of CD28 T cells and cumulative DEI

A good correlation was found between the percentage of CD28 lymphocytes and the cumulative DEI score (r = 0.51, P = 0.01) as well as between this score and the percentage of CD28 cells of CD8+ (r = 0.61, P = 0.007; Fig. 4). No correlation was seen between cumulative DEI score and the percentage of CD28 cells of CD4+ cells (r = 0.08, P = 0.7). Furthermore, there was a significant correlation between this score and the percentage of CD4+ lymphocytes (r = − 0.61, P = 0.002) and CD8+ lymphocytes (r = 0.61, P = 0.006) in whole blood, indicating a more severe disease course in patients with higher CD8 and lower CD4 lymphocytes. The positive correlation between the CD4/CD8 ratio and the cumulative DEI was r = 0.71 (P = 0.002).

Fig. 4
Correlation between CD28/CD8+ lymphocytes and the cumulative Disease Extent Index (DEI).

Lack of correlation between disease activity or therapeutic response and expression of costimulatory molecules

The expression of costimulatory molecules on both freshly isolated or in vitro stimulated B cells and monocytes showed no correlation with any of the clinical findings.

A comparison of active and inactive disease states disclosed no significant differences in the expression of costimulatory molecules (CD28, CTLA-4, B7-1, B7-2) (Table 2), either in vitro or in vivo, or on T cells, B cells and monocytes before and after stimulation. Moreover, the type of therapy had no effect on this expression.

Table 2
The expression of costimulatory molecules in percentage of CD3+, CD4+, CD8+, CD14+ and CD19+ cells after in vitro activation is given for active WG and WG in remission. None of the observed differences were significant

DISCUSSION

The data presented in this study reveal an unusual expression pattern of costimulatory molecules in patients with WG. The main finding is that CD28 T cells are more prevalent in WG patients than in HC. The frequency of these cells did not correlate with disease state and CD28 was not up-regulated on T cells after in vitro stimulation. Furthermore, the different therapies (i.e. prednisolone, cyclophosphamide and methotrexate) did not affect the CD28 cell frequency. Together with the bimodal frequency distribution in HC and WG patients, these findings indicate that CD28 cells are not an epiphenomenon of the disease but might instead be under the regulatory control of genetic or environmental factors and may predispose to WG. Comparable observations were made by Martens et al. [5] in patients with RA. They demonstrated a clear bimodal distribution of CD4+CD28 cells in both patients and controls and a higher frequency of this phenotype in the former. This condition was correlated with extra-articular disease manifestations. In the present study, organ involvement throughout the course of WG as measured by the cumulative DEI score was found to correlate with the percentage of CD28 lymphocytes. This indicates a more severe course of disease in individuals with a high frequency of CD28 T cells. There was a good correlation between this index and the percentage of CD8+/CD28 cells, whereas that of CD28of CD4+ cells did not correlate with any of the clinical or other laboratory findings, indicating that CD8+ cells play an important role in the pathogenesis of WG. Other recent data agree with these findings: in granulomatous lesions of WG patients we could demonstrate perforin using the in situ hybridization technique (unpublished data), while CD8+CD28 cells have been shown to produce perforin [12]. The further identification of these cells, NK or cytotoxic T cells, is in progress. CD8+CD28 cells were also found to exhibit high cytotoxic activity in vitro [13]. The very good correlation between the percentage of CD28+ lymphocytes and MFI of anti-CD28 indicates that individuals with low CD28+ cell counts also have a lower density of CD28 on these cells. On the whole, low expression of CD28 seems to be a risk factor for the development of more severe WG. Although Fagnoni et al. described an increase of CD8+CD28 cells in healthy ageing people [13], this effect can be ruled out in our patients, since they exhibited no correlation between CD8+CD28 frequency and age (r = 0.04, P = 0.86) or between CD8+CD28 frequency and disease duration (r = − 0.26, P = 0.62).

The significance of these cells in WG is not yet clear. It appears that CD28 T cells use other costimulatory pathways, as was demonstrated for these cells in RA [14]. Whether CD28 cells in WG use other costimulatory pathways remains to be clarified. Since CD28 costimulation promotes the production of Th2 cytokines [1517] and since the Th1 cytokine profile predominates in granuloma of WG patients [1], it is conceivable that a lack of CD28 augments the development of the Th1 pattern in WG. Even a direct effect on the cytokine profile is possible: CD8+CD28 cells have the ability to produce high amounts of IFN-γ, at least in HIV-infected individuals [18] in whom this cell type is also abundant [19,20]. In a previous study [1] we found that peripheral blood CD4+ cells and especially CD8+ cells produced IFN-γ. However, it remains to be clarified whether CD8+CD28 cells are the main source of IFN-γ in peripheral blood in WG. The possibility that the reduced CD28 expression in WG might be an epiphenomenon of the disease without pathogenic relevance can not yet be ruled out.

Another important finding of our study is the increased expression of B7-1 and B7-2 on T cells activated in vitro. The expression of B7-1 and B7-2 on these T cells is regulated by CD4+ cells in such a manner that addition of CD4+ cells prevents up-regulation, as demonstrated by Wolthers et al. [21]. The altered CD4/CD8 ratio in WG patients demonstrated in this and other reports [2,22] therefore might explain the increased expression of B7 ligands in vitro, which in our study was stronger on CD8+ than on CD4+ cells.

There is evidence in the literature that the expression pattern of costimulatory molecules is important for the development of the Th1 and/or Th2 reactions. In experimental allergic encephalomyelitis, blockade of the B7-1 pathway led to less severe disease and changed the cytokine profile from Th1 to Th2. In this model, B7-2 blockade enhanced the production of IFN-γ [23]. Comparable results were reported by Petro et al. [24]. In their study blockade of B7-2 depressed the production of IL-4. Furthermore, Th1 cells were found to depend more on B7 costimulation for their activation than did Th2 cells [25]. The increased expression of B7 on T cells and the decreased expression of B7-2 on monocytes in WG might therefore promote the Th1 immunoreaction leading to granuloma formation and necrotizing inflammation.

The increased expression of B7 ligands on T cells might also contribute to ongoing inflammation. Theoretically a self-costimulation of these cells is conceivable: B7+ CD4+ cell clones are able to stimulate resting peripheral blood T cells [26]. Therefore the enhanced stimulatory potential of these cells may contribute to persistently high levels of immune activation in WG.

The expression patterns of costimulatory molecules in WG patients resemble those found in HIV+ patients: decreased expression of CD28 leading to a higher percentage of CD4+CD28 and CD8+CD28 cells in WG patients (and HIV+ patients) versus HC, as well as an unusual increased expression of B7 ligands on T cells after in vitro activation. Furthermore, as in HIV-infected patients, the CD4/CD8 ratio in WG patients is decreased and correlates with more extended disease. The present study therefore may indirectly support the hypothesis of an infectious (viral?) aetiology of WG. However, further investigations are necessary to define the functional role of costimulatory molecules in the pathogenesis of WG and their impact on the still unknown aetiology of this disease.

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