David P. Andrew

α4β7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1

The mucosal vascular addressin, MAdCAM-1, is an immunoglobulin superfamily adhesion molecule for lymphocytes that is expressed by mucosal venules and helps direct lymphocyte traffic into Peyers patches (PP) and the intestinal lamina propria. We demonstrate that the lymphocyte integrin alpha 4 beta 7, also implicated in homing to PP, is a receptor for MAdCAM-1. Certain antibodies to alpha 4 and beta 7 integrin chains but not to the beta 2 integrin LFA-1 inhibit lymphocyte binding to purified MAdCAM-1 and to MAdCAM-1 transfectants. Lymph node lymphocytes, alpha 4 beta 7+ TK1 lymphoma cells, and a beta 7-transfected variant of an alpha 4+ B cell line, 38C13, bind constitutively to MAdCAM-1. Binding is enhanced by Mn(++)-induced integrin activation. The related integrin alpha 4 beta 1 supports efficient binding to VCAM-1 but not to MAdCAM-1, even after integrin activation, indicating that MAdCAM-1 is a preferential ligand for alpha 4 beta 7. Alpha 4 beta 7 can also bind VCAM-1, but this requires greater integrin activation than binding to MAdCAM-1. The findings imply a selective role for the interaction of alpha 4 beta 7 and MAdCAM-1 lymphocyte in homing to mucosal sites.

The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells

Lymphocytes that are responsible for regional (tissue-specific) immunity home from the blood to the intestines, inflamed skin or other sites through a multistep process involving recognition of vascular endothelial cells and extravasation. Chemoattractant cytokine molecules known as chemokines regulate this lymphocyte traffic, in part by triggering arrest (stopping) of lymphocytes rolling on endothelium. Here we show that many systemic memory T cells in blood carry the chemokine receptor CCR4 (ref. 6) and therefore respond to its ligands, the chemokines TARC and MDC. These cells include essentially all skin-homing cells expressing the cutaneous lymphocyte antigen and a subset of other systemic memory lymphocytes; however, intestinal (α4β7+) memory and naive T cells respond poorly. Immunohistochemistry reveals anti-TARC reactivity of venules and infiltration of many CCR4+ lymphocytes in chronically inflamed skin, but not in the gastrointestinal lamina propria. Moreover, TARC induces integrin-dependent adhesion of skin (but not intestinal) memory T cells to the cell-adhesion molecule ICAM-1, and causes their rapid arrest under physiological flow. Our results suggest that CCR4 and TARC are important in the recognition of skin vasculature by circulating T cells and in directing lymphocytes that are involved in systemic as opposed to intestinal immunity to their target tissues.

Rules of chemokine receptor association with T cell polarization in vivo

Current concepts of chemokine receptor (CKR) association with Th1 and Th2 cell polarization and effector function have largely ignored the diverse nature of effector and memory T cells in vivo. Here, we systematically investigated the association of 11 CKRs, singly or in combination, with CD4 T cell polarization. We show that Th1, Th2, Th0, and nonpolarized T cells in blood and tissue can express any of the CKRs studied but that each CKR defines a characteristic pool of polarized and nonpolarized CD4 T cells. Certain combinations of CKRs define populations that are markedly enriched in major subsets of Th1 versus Th2 cells. For example, although Th0, Th1, and Th2 cells are each found among blood CD4 T cells coordinately expressing CXCR3 and CCR4, Th1 but not Th2 cells can be CXCR3(+)CCR4(-), and Th2 but only rare Th1 cells are CCR4(+)CXCR3(-). Contrary to recent reports, although CCR7(-) cells contain a higher frequency of polarized CD4 T cells, most Th1 and Th2 effector cells are CCR7(+) and thus may be capable of lymphoid organ homing. Interestingly, Th1-associated CKRs show little or no preference for Th1 cells except when they are coexpressed with CXCR3. We conclude that the combinatorial expression of CKRs, which allow tissue- and subset-dependent targeting of effector cells during chemotactic navigation, defines physiologically significant subsets of polarized and nonpolarized T cells.

The Role of Thymus-Expressed Chemokine and Its Receptor CCR9 on Lymphocytes in the Regional Specialization of the Mucosal Immune System

Chemokines play an important role in the migration of leukocytes at sites of inflammation, and some constitutively expressed chemokines may direct lymphocyte trafficking within lymphoid organs and peripheral tissues. Thymus-expressed chemokine (TECK or Ckβ-15/CCL25), which signals through the chemokine receptor CCR9, is constitutively expressed in the thymus and small intestine but not colon, and chemoattracts a small fraction of PBLs that coexpress the integrin α4β7. Here we show that TECK is expressed in the human small bowel but not colon by endothelial cells and a subset of cells in intestinal crypts and lamina propria. CCR9 is expressed in the majority of freshly isolated small bowel lamina propria mononuclear cells (LPMC) and at significantly higher levels compared with colonic LPMC or PBL. TECK was selectively chemotactic for small bowel but not colonic LPMC in vitro. The TECK-induced chemotaxis was sensitive to pertussis toxin and partially inhibited by Abs to CCR9. TECK attracts predominantly the T cell fraction of small bowel LPMC, whereas sorted CD3+CCR9+ and CD3+CCR9− lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Collectively, our data suggest that the selective expression of TECK in the small bowel underlie the homing of CCR9+ intestinal memory T cells to the small bowel rather than to the colon. This regional specialization implies a segregation of small intestinal from colonic immune responses.

Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines

STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an α (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4+ T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19+ B cells and CD14+ monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.

C-C Chemokine Receptor 4 Expression Defines a Major Subset of Circulating Nonintestinal Memory T Cells of Both Th1 and Th2 Potential

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4+ thymocytes and on a major fraction of blood nonintestinal (α4β7−) memory CD4 lymphocytes, including almost all skin memory CD4+ cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4+ memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4+ CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4+ memory CD4 lymphocytes (including both CLA+ and CLA− fractions) readily express the Th1 cytokine IFN-γ after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.

Expression of Chemokine Receptors by Lung T Cells from Normal and Asthmatic Subjects

The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and α4β7. No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.

CCR4 in human allergen-induced late responses in the skin and lung

We studied the regulation of CCR4 expression in peripheral blood and in human models of cutaneous and pulmonary allergen challenge. CCR4 expression was detectable on freshly isolated CD4+ lymphocytes and in CD4+ and CD8+ T cell lines derived from blood of atopic donors. Numbers of CCR4+ cells were up‐regulated in T cell lines expanded in the presence of IL‐4. CCR4 mRNA was absent at baseline in normal subjects in lung and skin, but present at baseline in the lung of some atopics. Baseline expression of CCR4 mRNA and protein was higher in lung vs. skin, but allergen‐induced increases in CCR4 mRNA+ cells were observed in both organs. CCR4 protein+ cells were present at higher levels after allergen challenge in atopics compared to normal subjects. CCR4 may be important in the recruitment of T lymphocytes at sites of allergic inflammation, in a non‐organ‐specific manner.

CCR4 blockade does not inhibit allergic airways inflammation

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen‐challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage‐derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen‐specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.

9α,11β-PGF2 and its stereoisomer PGF2α are novel agonists of the chemoattractant receptor, CRTH2

CRTH2 is a recently described chemoattractant receptor for the prostaglandin, PGD2, expressed by Th2 cells, eosinophils and basophils, and believed to play a role in allergic inflammation. Here we describe the potency of several PGD2 metabolites at the receptor to induce cell migration and activation. We report for the first time that the PGD2 metabolite, 9α,11β‐PGF2, and its stereoisomer, PGF2α, are CRTH2 agonists. 9α,11β‐PGF2 is a major metabolite produced in vivo following allergen challenge, whilst PGF2α is generated independently of PGD synthetase, with implications for CRTH2 signalling in the presence or absence of PGD2 production.