Original paper
Increased expression of selected very late antigen integrin subunits on CD4 and CD8 blood T lymphocytes in patients with clinically stable asymptomatic atopic asthma

Introduction

Bronchial asthma is a chronic, inflammatory disease of the airways, which due to its frequency, poses an important public health problem. It is characterized by reversible airflow limitation and bronchial hyper-responsiveness; both leading to the distinct clinical symptoms: chest tightness, cough and breathlessness. Unfortunately, pathogenesis of asthma is only partially understood. It is associated with environmental factors together with variable inflammatory cell activation. T helper lymphocytes, in particular Th2 subtype are likely to be pivotal in directing the disease development and progression and leading to the eosinophilic airway inflammation. Activated Th2 cells are present in the airways even when the disease is quiescent. Adoptive transfer of Ag-primed T cells in naïve animals induces eosinophilia, bronchial hyper-responsiveness and late airway response [1]. Eosinophils are considered the major effector cells in asthmatic airway inflammation. Interestingly, anti-IL-5 antibodies, able to reduce blood and lung eosinophilia, did not change asthma severity [2]. Some believe that non-eosinophilic inflammation and airway remodelling contribute to the persistence and progression of the disease, despite anti-inflammatory therapy [3].

Recruitment of the engaged cells is crucial to the development of the inflammatory reaction. This process is mediated by adhesive molecules, such as glycoproteins, selectins and integrins [4]. Integrins are a family of heterodimeric glycoproteins composed of non-covalently associated a and β subunits. They are present on almost all viable cells and regulate between cell and cell to matrix interactions [5]. Also leukocyte migration into the lung is mediated by integrins, in particular those containing a4 (a41 and a47) and 2 subunits (LFA-1 – integrin aL2 – CD11a/CD18; Mac-1 – integrin aM2 – CD11b/CD18, integrin aX2 – CD11c/CD18 and integrin aD2 – CD11d/CD18). All those integrins have been intensively studied in inflammatory diseases, like asthma, and considered a possible therapeutic target [6, 7]. We have previously suggested that collagen integrin receptors: a11 and a21, both found on peripheral blood eosinophils of asthmatic subjects, may be involved in asthma pathogenesis [8]. The a11 integrin belongs to the 1 inte­grin family (also called very late antigen-1 – VLA-1). It is a specific receptor for collagen IV, the main component of the small vessel basement membrane [9]. Very late antigen-1 is expressed inter alia on fibroblasts, mesenchymal and epithelial cells, macrophages and, NK and T lymphocytes after their activation [10]. It plays a role in angiogenesis, metastasis, cell migration and cytokine secretion. Anti-inflammatory effects of the VLA-1 antagonists were observed in animal models of arthritis [11], colitis [12], allergen-induced bronchoconstriction [13] and glomerulonephritis [14].

The a21 integrin (VLA-2) is a collagen I receptor. Its main function is connected with haemostasis. VLA-2 also promotes neoplasm invasion [15]. But collagen I is also an important component of extracellular matrix of the lungs and VLA-2 was described as a stimulator of collagen and fibronectin accumulation in the airways – crucial element of airway remodelling [16, 17].

Aim

The aim of this study was to analyse the expression of collagen receptors: integrins a11 and a21 on peripheral blood CD4 and CD8 T lymphocytes in subjects with chronic clinically stable atopic asthma.

Material and methods

The study was conducted on 25 adult atopic asthmatics and 17 healthy controls. All asthma patients were in a stable clinical condition with mild (n = 15) to moderate (n = 10) persistent asthma, according to the GINA guidelines [18]. Their atopic status was confirmed by a positive skin testing for at least one standard inhaled allergen (Allergopharma, Germany). Most of them had a good or partially controlled asthma (GINA 2006) and were treated with a medium dose of inhaled glucocorticosteroids and long-acting 2-agonists. Smokers and patients suffering from heart failure, diabetes mellitus, renal or hepatic diseases, as well as other chronic diseases were excluded from the study. The control group consisted of non-atopic and non-smoking volunteers, without any chronic illness. Our study was approved by the Jagiellonian University Ethical Committee and all subjects gave informed consent to participate in this study.

Expression of a1, a2, a4 and 1 integrin subunits on CD4 and CD8 peripheral blood T lymphocytes was analysed by flow cytometry (Epics XL, Beckman Coulter International, Nyon, Switzerland) and expressed as Median Fluorescence Intensity (MFI).

A 100 µl samples of venous peripheral blood taken on EDTA was incubated with monoclonal antibodies (mAbs) in a concentration of 10 µl/100 µl using threefold staining:

1) PerCP-conjugated mouse anti human-CD3 mAb for lymphocyte T detection;

2) FITC-conjugated mouse anti human-CD4 or CD8 mAb;

3) PE-conjugated mouse anti human – CD49a (anti-a1), or CD49b (anti-a2), or CD49d (anti-a4), or CD29 (anti-1) mAbs.

All monoclonal antibodies were purchased from BD Pharmingen, (San Diego, CA, USA). The isotypic control was performed using mouse IgG antibody against keyhole limpet hemocyanin antigen (KLH), purchased from Becton Dickinson Biosciences (San Jose, CA, USA) and PE, FITC

and PerCP – labelled goat anti-mouse IgG (1 µg/100 µl; Jackson Immuno Research Laboratories, West Grove, PA, USA). Serum concentration of IgE and eosinophil cationic protein (ECP) was measured using UniCAP System, Pharmacia, Sweden.



Statistical analysis



Normally distributed results were reported in tables as a mean ± standard deviation (SD). Values, which were not normally distributed, were presented in tables as a median with interquartile range and in figures as a median with standard error of median. Comparison between experimental groups were tested using Mann-Whitney U test, whilst relationships were tested using Spearman Rank Correlations. Two-sided 5% level of significance was used. All statistical testing was performed by Statistica StatSoft (Tulsa, OK, USA) software.

Results

The summary of the results is shown in Tables 1, 2 and Figures 1 and 2. The groups studied were similar as to age and sex. Patients had significantly higher blood eosinophilia and serum levels of ECP and IgE (Table 1).

As shown in Table 2 and Figure 1, the expression of both a4 and 1 chains was significantly higher in asthma subjects as compared to controls, but only for CD4

T cells (p = 0.02 and p = 0.0004, respectively). The a1 subunit was absent in almost all subjects studied (only a few asthma patients had detectable fluorescence for a1 chain). On the other hand, a2 subunit was found on blood T lymphocytes of both studied groups, but its expression was low, slightly higher in asthma, particularly on CD4 T cells (the difference was not significant).

Surprisingly, in subjects suffering from asthma for longer than 4 years (n = 15) not only a4 and 1, but also a2 chain was overexpressed on CD4 T cells, with a2 and a4 overexpression on CD8 (Figure 2).

Similarly, a significantly higher expression of a2, a4 and 1 was observed on CD4 T cells in a group of patients with at least one asthma exacerbation during the last 12 months, in comparison to healthy subjects (n = 9, p = 0.04; p = 0.04; p = 0.001, respectively).

No correlation was found between the chain expression, dose of inhaled steroids, IgE level, ECP or eosino­phils count. A positive correlation was found for blood eosinophilia and ECP concentration (p = 0.009) only.

Discussion

An increased expression of a4 and 1 subunits on blood CD4 T lymphocytes in clinically stable, asymptomatic asthmatics confirmed pre-activation and readiness for migration of these cells to the inflammatory site. However, in our studies, both chains were also detected on blood lymphocytes in healthy individuals and on peripheral blood eosinophils in healthy and asthmatic subjects [8]. Both are intensively studied as a possible therapeutic target in many inflammatory diseases, such as asthma [6, 7, 19]. Integrins containing a4 and 1 subunits are important or even critical for immunity and body health control. For this reason blockade of a4 and 1 could be hazardous and lead to unexpected outcomes. Natalizumab, a humanized IgG4 anti-a4-integrin monoclonal antibody inhibits both: a47/mucosal addressin-cell adhesion molecule-1 (MadCAM-1) and a41/vascular-cell adhesion molecule-1 (VCAM-1) interactions. Administration of natalizumab was shown to be highly effective in patients with multiple sclerosis and Crohn’s disease [20, 21]. Unfortunately, it has been also implicated in some cases of progressive multifocal leukoencephalopathy, due to JC virus activation as a consequence of severe immunosuppression [22]. It seems that for therapeutic purposes, a target more selective and specific to a particular disease could be safer. In our study, an expression of a2 subunit on blood lymphocytes was low, but increased in patients suffering from asthma for longer than 4 years, even despite chronic anti-inflammatory therapy and lack of clinical symptoms. Our findings were not in a clear relationship with the type of treatment and current severity of symptoms. The biological function of a21 in asthma is still unknown. An increased expression of this integrin was found by others on blood lymphocytes but only during severe exacerbation of asthma [23, 24]. We also found a higher expression of a2 subunit on CD4 T cells in patients, who had at least one exacerbation during the last year of the patient’s observation. We speculate that integrin a21 is not involved in lymphocyte transmigration but rather acts on turnover of the extracellular matrix, as a stimulator of remodelling. The a21 integrin has been described previously as a stimulator of collagen and fibronectin accumulation in the airways [25]. Fibroblasts initiate collagen degradation through this integrin [26], whereas a11 integrin is important in collagen fibrils organization and in feedback inhibition of collagen I synthesis [27]. Our findings indicate that an expression of 2 on T cells is much lower than this observed for e.g. 4 and b1. In our preliminary experiments with transmigration of eosinophil and lymphocyte through human extracellular matrix and collagen I coated inserts, blockade of a2 integrin subunit by functional active mAbs, as well as

VP-12 (viper venom lectin – selective inhibitor of a2b1 integrin) had no impact on lymphocyte transmigration, but decreased migration of eosinophils (data not published).

Till now airway remodelling could not be reversed. Current recommended asthma treatment is partially successful in limiting allergic inflammation but does not specifically address the remodelling process. Despite aggressive treatment asthma often progresses [28]. Clinical studies suggest that use of inhaled glucocorticoids before the age of 2 has no effect on asthma 8 years later [29]. Experimental asthma therapy with imatinib (tyrosine kinase inhibitor) can prevent airway inflammation and remodelling in the murine model by inhibition of collagen deposition [30, 31]. The mode of such action is unknown, but could be connected to interaction with integrin collagen receptors. Integrin-linked cytoplasmic kinase can activate and regulate smooth muscle contraction directly by myosin phosphorylation and indirectly by myosin light-chain phosphatise inhibition [32]. There are only five known collagen integrin receptors: a11, a21, a31, a101 and a111 [33]. We studied two of them, but it is tempting to speculate that integrin collagen receptors could become a new therapeutic target in treatment of asthmatic airway remodelling.

Acknowledgments

This work was supported by KBN – the State Committee for Scientific Research: registration number N N402 186835.

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