Cxcr3 and its ligand CXCL10 are expressed by inflammatory cells infiltrating lung allografts and mediate chemotaxis of T cells at sites of rejection

Abstract

The attraction of T lymphocytes into the pulmonary parenchyma represents an essential step in mechanisms ultimately leading to lung allograft rejection. In this study we evaluated whether IP-10 (CXCL10), a chemokine that is induced by interferon-gamma and stimulates the directional migration of activated T cells, plays a role in regulating the trafficking of effector T cells during lung allograft rejection episodes. Immunohistochemical examination showed that areas characterized by acute cellular rejection (grades 1 to 4) and active obliterative bronchiolitis (chronic rejection, Ca) were infiltrated by T cells expressing CXCR3, i.e., the specific receptor for CXCL10. In parallel, T cells accumulating in the bronchoalveolar lavage of lung transplant recipients with rejection episodes were CXCR3+ and exhibited a strong in vitro migratory capability in response to CXCL10. In lung biopsies, CXCL10 was abundantly expressed by graft-infiltrating macrophages and occasionally by epithelial cells. Alveolar macrophages expressed and secreted definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the secretory capability of alveolar macrophages was up-regulated by preincubation with interferon-gamma. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the bronchoalveolar lavage in individuals with rejection episodes. These data indicate the role of the CXCR3/CXCL10 interactions in the recruitment of lymphocytes at sites of lung rejection and provide a rationale for the use of agents that block the CXCR3/CXCL10 axis in the treatment of lung allograft rejection.

Figure 1.

Immunohistochemistry for CXCR3 expression by lung T cells infiltrating transbronchial biopsy of four representative allograft recipients. a and b: Two allograft recipients with a rejection episode classified as A2 and A3 grades, respectively. c and d: Two representative lung allograft recipients with active bronchiolitis obliterans. Strongly stained CXCR3+ lymphocytes can be demonstrated not only in the perivascular space but also in the alveolar septa and air spaces of patients with acute cellular rejection (a and b). In patients with active obliterative bronchiolitis (c and d) subepithelial fibrosis can be observed in the bronchiole and is associated with a moderate infiltrate of CXCR3 stained lymphocytes. Original magnification, ×400.

Figure 2.

The flow cytometry profile of BAL T cells recovered from three representative patients with A1-A3 rejection episodes, a patient with active BOS, a patient showing no rejection signs, and a control patient. The expression of CXCR3 and cytoplasmic cytokines has been determined as reported in the Materials and Methods section. The figure shows that BAL T cells from lung allograft recipients with A1-A3 rejection episodes or BOS express CXCR3 and IFN-γ but not IL-4. CXCR3 was expressed at higher intensity by BAL T cells of patients with higher grade acute rejection (A2-A3) or BOS than in the patients with the lower grade rejection (A1) (P > 0.01 at the Kolmogorov-Smirnov analysis).

Figure 3.

Chemotactic activity of CXCL10 on BAL T cells purified from three representative patients with A1-A3 rejection episodes and a patient with active BOS. The assays were performed using a modified Boyden chamber in triplicate and data are given as mean ± SD. CXL10 shows significant chemotactic activity on BAL T cells (A–D) and the CXCR3+ T cell clone (E). The number of BAL T cells migrating in response to CXCL10 was higher in patients with A2-A3 grade rejection than in patients with lower grade rejection (A1). Because of the limited number of patients evaluated the difference was not significant.

Figure 4.

Immunohistochemistry for CXCL10 expression by AMs infiltrating transbronchial biopsy in a representative lung allograft recipient with a rejection episode classified as grade A2. The figure shows that several AMs are strongly marked by the anti-CXCL10 antibody. Scattered epithelial cells also bear CXCL10. Original magnification, ×400.

Figure 5.

Flow cytometry profile of AMs recovered from three representative patients with A1-A3 rejection episodes, a patient with active BOS, a patient showing no rejection signs, and a control patient. The expression of cytoplasmic cytokines was determined as reported in the Material and Methods section. The figure shows that AMs from lung allograft recipients with A1-A3 rejection episodes or BOS express CXCL10.

Figure 6.

Chemotactic activity of supernatants obtained from 24-hour cultures of AMs from three representative patients with A1-A3 rejection episodes and a patient with active BOS. Supernatants obtained from AMs of lung allograft recipients with rejection episodes or with BOS exerted chemotactic activity on the CXCR3 + 300-19 T-cell line that was inhibited by a neutralizing anti-CXCL10 antibody (white bars). The inhibitory activity was not complete, indicating that AMs release other proteins into supernatants that are able to interact with the CXCR3+ T cell clone.

Figure 7.

The fluid components of the BAL obtained from lung allograft recipients with rejection episodes or with BOS exert chemotactic activity on the CXCR3 + 300-19 T cell line, indicating the presence of CXCR3 ligand(s). For a comparison, the figure shows the migratory response of the 300-19 T cell line in response to rhCXL10 (white bars). The assays were performed in triplicate, and data are given as mean ± SD.