The Identification, Characterization, and Distribution of Guinea Pig CCR4 and Epitope Mapping of a Blocking Antibody

Abstract

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon ␥. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on ϳ12% of guinea pig peripheral blood T helper cells, and CCR4 ؉ guinea pig thymocytes were detected in low numbers. However, CCR4؉ T helper cells constituted ϳ9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.Allergic inflammation of the lung in diseases such as asthma is largely controlled and maintained by the recruitment of antigen-specific T lymphocytes to the lung tissue (1). The Th2-type lymphocytes that predominate at sites of allergic inflammation secrete cytokines such as IL-4, IL-5 and IL-13, 1 which influence the actions of other leukocyte populations involved in the inflammatory response (2, 3). For example, IL-4 and IL-13 can induce B lymphocyte production of IgE (4, 5) and airway epithelial cell generation of eotaxin, which is central to eosinophil recruitment (6, 7), whereas IL-5 is a potent eosinophil maturation and survival factor (8, 9). Overexpression of IL-13 in the lung causes many of the histopathological and physiological features of asthma (10). Increasing evidence suggests that chemokines are important regulators of the selective trafficking of leukocytes in homeostasis, hematopoiesis, and inflammation (11). Chemokines can be divided into four subfamilies dependent upon the position of their amino-terminal cysteine residues; the two main chemokine groups are the CXCL and the CCL subfamilies (12), which signal via CXCR and CCR receptors belonging to the G-protein coupled receptor superfamily (13,14). The differential expression of chemokine receptors on the surface of leukocytes contributes to the selective recruitment of cells to specific sites, and, consequently, these receptors may provide useful targets for therapeutic intervention in the modulation of inflammatory disorders. Studies of T lymphocytes polarized in vitro have shown that Th1 cells preferentially express CC...

CCR4 blockade does not inhibit allergic airways inflammation

“…We observed moderately higher proportions of Th cells expressing CCR4 in the blood than in our previous work [17] and found CCR4 on a higher proportion of lung Th + cells. The increased proportion of Th + cells that were CCR4 + in the lungs in these experiments may be a consequence of sensitization or perhaps reflects increased sensitivity of staining in these experiments.…”

“…Guinea pig homologues of CCL22 and CCL17 have not yet been identified; thus, analysis of mRNA expression and development of specific ELISAs for guinea pig CCR4 ligands are not yet possible. We tried to inhibit the recruitment of CCR4 + cells by treatment with an anti-CCR4 mAb at a dose predicted to block the receptor in vivo, based on our previous studies showing effective blockade of CCR4 at concentrations of 20 μg/ml in vitro [17]. Our data show that administration of the anti-CCR4 mAb caused persistent labeling of CCR4 in vivo over 48 h and that further staining of blood from these animals with anti-CCR4 in vitro failed to identify a population of cells that was not already bound by antibody, suggesting effective blockade was likely to be maintained during the course of study.…”

“…The immunoglobulin G (IgG)2a isotype anti-CCR4 mAb 10E4 [17] was prepared in-house at Millennium Pharmaceuticals Inc. (Cambridge, MA), and endotoxin < 1 EU/ml. Sterile mouse IgG was from Jackson Laboratories (West Grove, PA); R-phycoerythrin (RPE)-rabbit anti-mouse IgG, from Dako (Ely, UK); fluorescein isothiocyanate (FITC)-anti-guinea pig Th and control Ab, from Serotec (Oxford, UK).…”

“…Aliquots were sequentially incubated at 4°C with buffer, 10E4, or isotype control (10 μg/ml) for 30 min, RPE secondary Ab (preabsorbed with 10% guinea pig serum) for 20 min, mouse IgG (50 μg/ml) for 10 min, and FITC anti-guinea pig Th Ab (1:25 dilution) for 30 min as described [17]. Red cells were lysed with FACSLyse (Becton Dickinson, Cowley, UK) and analyzed by FACS.…”

“…Similarly, studies of CCR8 −/− mice mostly fail to demonstrate a significant contribution of this receptor to allergic pulmonary inflammation [13,14], although one study demonstrated a role for CCR8 in allergic inflammation [15], and another has shown a role for the CCR8 ligand, CCL1, in eosinophil (but not Th2 T cell) trafficking [16]. We have previously identified guinea pig CCR4, showed it responded to human and murine CCR4 ligands, and described a monoclonal antibody (mAb) that blocked CCR4 signaling on guinea pig T cells in vitro [17]. Here, we investigated the actions of this mAb on allergic inflammatory responses in the guinea pig lung.…”