T Cells Activation and Interferon-γ/Nitric Oxide Production during Behçet Disease: A Study in Algerian Patients

Abstract

The aim of the current study is to situate CDs expression (CD3+, CD4+, CD8+) and their relationship with IFNγ and NO production in Algerian patients with Behçet disease (n = 34 ). Our results revealed an elevated expression of T cells markers tested in patients in active stage of the disease in relation to IFNγ and NO increase levels. Most interestingly, we noted that CD4+/CD8+ expression correlated with IFNγ and NO production in active stage. In contrast, we observed a decrease of these mediators in inactive stage. Collectively, our results indicate a dependent relation between these parameters and inflammatory response associated with the evolutionary stage of the disease.

Keywords::

• Behçet’s disease
• CD
• interferon gamma
• nitric oxide
• cytokine

Behçet’s disease (BD) is an inflammatory multisystemic disorder involving mucocutaneous, ocular, arthritic, vascular and central nervous systems The diagnosis is clinical and based on international criteria.¹ It is most prevalent in the Mediterranean countries, including Algeria, and along the Silk Route.²

The involvement of cytokines and chemokines in BD pathogenesis is reflected by the increase of their concentrations in sera and in PBMC of patients with BD and some of these mediators correlate with the clinical activity of the disease.^2,3 The study of CDs expression, as a markers of Tcells in relation with IFNγ production involved in the immune responses during BD appears necessary to situate Th1 immune response in the evolution and may be in the genesis of BD.^4,5 IFNγ is a pivotal Th1 cytokine triggered NO synthase 2induction.^2,3 Several works reported its production during the Behçet disease.^3,4 We report here the study of CD3,CD4, CD8 expression in BD Algerian patients in relation with IFNγ and NO production. Our observations led us to investigate the involvement of Tcell activation in inflammatory process enhanced in the disease.

Algerian Behçet patients (20 men and 14 women; mean age 27.65 ± 13.57 years; range 23–59 years) were classified into two groups of patients according to the evolutionary stage of the disease.

The patients tested in our current study were hospitalized in the internal medicine department at Ain Naadja hospital, Algiers, Algeria. All patients responded to the ISG criteria for Behçet’s Disease.¹

The patients were followed during two years. In the beginning of our investigations, all the patients (n = 34) were in the active phase. Twenty five (n = 25) of them had severe disease and were classified into the active stage group. These patients did not responded to the therapy and were in the major times in pushes phases, or passed a short period of remission stage(between 4 and 6 weeks), they became rapidly in releapse stage. In the same group of the 34 patients, 9 subjects became in remission stage after 8 months of therapy and they did not releapse along our study. These subjects were classified into inactive stage group of Behçet disease

The patients in active stage (PA) were tested in the beginning of therapy (corticoids: prednisone) and every month along our study (2 years). The patients in inactive stage (PI) were tested with minimal therapeutic dose of corticoids, or without any therapy. Healthy controls (n = 35) were obtained from adult volunteer donors. All participants gave informed consent for the present study, which was carried out according to the guideline of the local Ethics Working Group. CDs determination was assessed using flow cytometric analysis. IFNγ evaluation in the sera and in the supernatants of PBMC cultures from patients stimulated by phytohaemagglutinin (PHA) was assessed by ELISA method and NO production by modified Griess method.⁶

All results were expressed as mean standard deviation. Data analysis was performed using Minitab 15. Student’s t-test was used for comparison between different groups. Differences were considered to be statistically significant at P < 0.05.

Our results revealed an increased expression of Tcells markers tested in patients in active stage of the disease in relation to IFNγ and NO increase levels assessed in sera and in supernatant of PBMC cultures of patients. Most interestingly, we noted that CD4/CD8/CD3 expression correlated with IFNγ and NO production in active stage (Table 1). In contrast, we have observed a decrease of these mediators in inactive stage both in vivo and ex vivo in PBMC of patients stimulates by PHA (Table 1).

TABLE 1  Assessment of Tcells markers and IFN-γ/NO in vivo and ex vivo in Algerian patients with Behçet disease.

Patients n = 34	CD4%	CD8%	CD3%	IFN-γ in serum UI/ml	NO in serum µM	IFN-γ in PBMC UI/ml	NO in PBMC µM
Patients in active stage PA n = 25	69 ± 14.18	51.81 ± 8.15	90.74 ± 4.88	36 ± 3.4	163 ± 46.8	61.9 ± 9.3	267.54 ± 19.27
Patients in active stage PI n = 9	47.33 ± 7.76	23 ± 2.19	70.08 ± 3.51	13.6 ± 6.3	89.17 ± 21.1	38 ± 7.8	173.71 ± 26.10
Heathy controls n = 35	40.76 ±2.09	17.2 ± 5.74	68.64% ± 4.87	4.6 ± 1.9	73.6 ± 12.4	19.6 ± 8.1	98.76 ± 39.41

-CD4,CD8,CD3 expression, IFN-γ and NO levels in vivo and ex vivo in Algerian patients with Behçet disease (PA, n = 25; PI, n = 9) and healthy controls (n = 35). Values represent mean ± standard deviation. Significance compared with healthy donors (P < 0.05). Significant difference between the two groups of Behçet patients (P < 0.01).

The high expression of CD3 in patients in active stage, suggests a strong T cell activation in BD (Table 1).Corticosteroids therapy used is responsible of the decline of CD4 and CD8 expression, in particular in inactive stage (Table 1).^7,8

Our data indicate that both CD4 and CD8 T cells subset could play a key role in the pathogenesis of BD, probably by their ability to produce IFN-γ. This hypothesis is supported by strong relationship observed between T cell activation IFN γ levels in vivo and in vitro system (Table 1).

In BD, several authors reported that chronic inflammation is CD8 dependent and related to the lack of Fas expression in activated LTCD4.^9,10 Our study is in the line with these data, considering CD8 elevation levels in active stage of the disease. Our results show the evident involvement of IFN γ in the inflammatory process accompanying Behçet pathology.^3,5

On the other hand, this study provides evidence that NO production is elevated in Behçet patients in active stage compared to the remission stage and the healthy controls. Our observations are in the line with those reported by many other authors showing the elevation of NO concentration in the sera of active Behçet patients.^2,3 Several studies have suggested that the overexpression of either inducible NO and proinflammatory cytokines might be intimately involved in the pathogenesis and the evolution of BD.^3,11

We suggest the possible involvement of both CD4 and CD8 Tcells in inflammatory process probably via IFN- γ induction knowing that this cytokine is highly required in up regulation of nitric oxide production by monocyte/macrophage system.

Considering the correlation between IFN-γ and NO levels, we postulate that IFNγ stimulates inducible NO Synthase activity in PBMC from Behçet patients and promotes inflammation through its ability to mediate the chemotaxis of both neutrophil and monocytes.^2,3 Collectively, our results indicate a dependent relation between T cells activation and inflammatory response associated with the evolutionary stage of the disease.

Declaration of interest: No competing financial interest exist.