Expression of the Chemokine Receptors CCR4, CCR5, and CXCR3 by Human Tissue-Infiltrating Lymphocytes
2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64378-7
ISSN1525-2191
AutoresEric J. Kunkel, Judie Boisvert, Kristine Murphy, Mark A. Vierra, Mark C. Genovese, Andrew J. Wardlaw, Harry B. Greenberg, Martin R. Hodge, Lijun Wu, Eugene C. Butcher, James J. Campbell,
Tópico(s)Immune Cell Function and Interaction
ResumoDifferential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4+ lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4+ populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4+ lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69+). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in α4β7 expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation. Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4+ lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4+ populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4+ lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69+). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in α4β7 expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation. Tissue-specific recruitment and retention of lymphocyte subsets compartmentalizes the immune system. Such partitioning increases the likelihood that lymphocytes bearing rare antigen specificities will encounter their cognate antigen. At the same time, compartmentalization decreases the likelihood that an expanded lymphocyte population (specific for an antigen found only within a given tissue) will be exposed to a potentially cross-reactive host-derived antigen within a different tissue.1Butcher EC Picker LJ Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2523) Google Scholar Tissue-targeted recruitment and localization are thought to be achieved by regulated expression of particular homing receptors on lymphocytes (including adhesion and chemokine receptors), as well as by regulation of their counter-receptors expressed by endothelial, epithelial, inflammatory, and parenchymal cells in the target tissues.2Butcher EC Williams M Youngman K Rott L Briskin M Lymphocyte trafficking and regional immunity.Adv Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar, 3Campbell JJ Butcher EC Chemokines in tissue-specific and microenvironment-specific lymphocyte homing.Curr Opin Immunol. 2000; 12: 336-341Crossref PubMed Scopus (559) Google Scholar The clearest evidence to support the concept of tissue-dedicated trafficking of peripheral blood lymphocyte subsets exists at the level of their expression of adhesion molecules.1Butcher EC Picker LJ Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2523) Google Scholar, 2Butcher EC Williams M Youngman K Rott L Briskin M Lymphocyte trafficking and regional immunity.Adv Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar Circulating lymphocytes associated with skin homing express the E-selectin ligand CLA (cutaneous lymphocyte antigen), whereas those associated with homing to the gastrointestinal tract express the MAdCAM-1 ligand α4β7 integrin. Although skin- and intestinal-specific lymphocyte populations are the best studied, there is reason to believe that additional populations dedicated to other tissues may exist.1Butcher EC Picker LJ Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2523) Google Scholar, 2Butcher EC Williams M Youngman K Rott L Briskin M Lymphocyte trafficking and regional immunity.Adv Immunol. 1999; 72: 209-253Crossref PubMed Google Scholar In addition to differential expression of adhesion molecules, differential expression of chemokine receptors may also contribute to tissue-specific homing. This hypothesis would predict the disproportionate expression of particular chemokine receptors by lymphocytes that have infiltrated tissues that express the corresponding chemokine ligand. We have recently found support for this prediction.4Kunkel EJ Campbell JJ Haraldsen G Pan J Boisvert J Roberts AI Ebert EC Vierra MA Goodman SC Genovese MC Wardlaw AJ Greenberg HB Parker CM Butcher EC Andrew DP Agace WW Lymphocyte CCR9 and epithelial TECK expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (548) Google Scholar By carefully isolating T cells from various human tissues, we demonstrated that the chemokine receptor CCR9 is highly expressed by nearly all T lymphocytes infiltrating the small intestine (the one organ outside of the thymus where the CCR9 ligand TECK is highly expressed). In contrast, CCR9 was not expressed by T lymphocytes isolated from other tissues tested, including skin, liver, synovium, and lung. In contrast to small intestine-derived lymphocytes, CCR9 was expressed by only a small percentage of colon lymphocytes, suggesting that circulating intestinal-specific lymphocytes may be further subdivided into small intestine-dedicated versus large intestine-dedicated populations.4Kunkel EJ Campbell JJ Haraldsen G Pan J Boisvert J Roberts AI Ebert EC Vierra MA Goodman SC Genovese MC Wardlaw AJ Greenberg HB Parker CM Butcher EC Andrew DP Agace WW Lymphocyte CCR9 and epithelial TECK expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (548) Google Scholar To further examine similarities and differences among lymphocytes that have infiltrated diverse human tissues, we have investigated the differential expression of three more chemokine receptors (CCR4, CCR5, and CXCR3) by tissue-infiltrating lymphocytes. We previously reported that circulating skin-associated CLA+ lymphocytes uniformly express high levels of CCR45Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar and that the CCR4 ligand TARC6Imai T Baba M Nishimura M Kakizaki M Takagi S Yoshie O The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4.J Biol Chem. 1997; 272: 15036-15042Crossref PubMed Scopus (473) Google Scholar is expressed on normal and inflamed cutaneous (but not intestinal) endothelium. Taken together, these two findings suggest that CCR4 and TARC may participate in skin-specific lymphocyte recruitment.3Campbell JJ Butcher EC Chemokines in tissue-specific and microenvironment-specific lymphocyte homing.Curr Opin Immunol. 2000; 12: 336-341Crossref PubMed Scopus (559) Google Scholar, 5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar Interestingly, CCR4 monoclonal antibodies (mAbs) also recognized a subset of CLA−/α4β7− peripheral blood memory CD4+ lymphocytes.5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar The role of CCR4 here is unknown, but these cells may represent another skin-associated subset or a subset dedicated to a systemic tissue. CCR5 and CXCR3 have been reported to be tissue-specific receptors in multiple organs. CCR5 is expressed by lymphocytes in intestinal tissues and was suggested to be a mucosa-specific receptor.7Agace WW Roberts AI Wu L Greineder C Ebert EC Parker CM Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation.Eur J Immunol. 2000; 30: 819-826Crossref PubMed Scopus (131) Google Scholar, 8Mackay CR Dual personality of memory T cells.Nature. 1999; 401: 659-660Crossref PubMed Scopus (54) Google Scholar However, other groups reported CCR5 expression on lymphocytes in the brains of multiple sclerosis patients,9Sorensen TL Tani M Jensen J Pierce V Lucchinetti C Folcik VA Qin S Rottman J Sellebjerg F Strieter RM Frederiksen JL Ransohoff RM Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients.J Clin Invest. 1999; 103: 807-815Crossref PubMed Scopus (901) Google Scholar liver,10Shields PL Morland CM Salmon M Qin S Hubscher SG Adams DH Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver.J Immunol. 1999; 163: 6236-6243PubMed Google Scholar and on synovial lymphocytes from arthritis patients.11Qin S Rottman JB Myers P Kassam N Weinblatt M Loetscher M Koch AE Moser B Mackay CR The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.J Clin Invest. 1998; 101: 746-754Crossref PubMed Scopus (1206) Google Scholar, 12Suzuki N Nakajima A Yoshino S Matsushima K Yagita H Okumura K Selective accumulation of CCR5+ T lymphocytes into inflamed joints of rheumatoid arthritis. Int.Immunol. 1999; 11: 553-559Google Scholar CXCR3 expression was similarly reported on intestinal,7Agace WW Roberts AI Wu L Greineder C Ebert EC Parker CM Human intestinal lamina propria and intraepithelial lymphocytes express receptors specific for chemokines induced by inflammation.Eur J Immunol. 2000; 30: 819-826Crossref PubMed Scopus (131) Google Scholar inflamed brain,9Sorensen TL Tani M Jensen J Pierce V Lucchinetti C Folcik VA Qin S Rottman J Sellebjerg F Strieter RM Frederiksen JL Ransohoff RM Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients.J Clin Invest. 1999; 103: 807-815Crossref PubMed Scopus (901) Google Scholar liver,10Shields PL Morland CM Salmon M Qin S Hubscher SG Adams DH Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver.J Immunol. 1999; 163: 6236-6243PubMed Google Scholar and synovial11Qin S Rottman JB Myers P Kassam N Weinblatt M Loetscher M Koch AE Moser B Mackay CR The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.J Clin Invest. 1998; 101: 746-754Crossref PubMed Scopus (1206) Google Scholar, 12Suzuki N Nakajima A Yoshino S Matsushima K Yagita H Okumura K Selective accumulation of CCR5+ T lymphocytes into inflamed joints of rheumatoid arthritis. Int.Immunol. 1999; 11: 553-559Google Scholar lymphocytes. Thus, the roles of CCR4, CCR5, and CXCR3 in tissue-specific lymphocyte homing require further clarification. By identifying the similarities and differences in expression of chemokine receptors by lymphocytes that have infiltrated a variety of extralymphoid tissues, we hope to gain an understanding of the specialized CD4+ lymphocyte subsets involved in inflammation and immune surveillance in particular tissue sites. Such knowledge may identify pharmacological targets that could be used to manipulate tissue-specific homing or other chemokine-mediated inflammatory functions, making possible treatments for regional autoimmune diseases (eg, psoriasis or Crohn's disease) as well as tailored treatments to prevent rejection of transplanted tissues. Anti-human CCR4 mAbs 1G1 and 2B10 (both mouse IgG1),5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar CLA-FITC mAb HECA-452 (rat IgM),13Berg EL Yoshino T Rott LS Robinson MK Warnock RA Kishimoto TK Picker LJ Butcher EC The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.J Exp Med. 1991; 174: 1461-1466Crossref PubMed Scopus (505) Google Scholar and α4β7-PE mAb Act-1 (mouse IgG1)14Schweighoffer T Tanaka Y Tidswell M Erle DJ Horgan KJ Luce GE Lazarovits AI Buck D Shaw S Selective expression of integrin alpha 4 beta 7 on a subset of human CD4+ memory T cells with hallmarks of gut-trophism.J Immunol. 1993; 151: 717-729PubMed Google Scholar have been previously described. Unconjugated mouse anti-human CD45RO (IgG2a, clone UCHL1), CCR5 (IgG2a, clone 2D7; known to correlate best with genetic expression of CCR515Hill CM Kwon D Jones M Davis CB Marmon S Daugherty BL DeMartino JA Springer MS Unutmaz D Littman DR The amino terminus of human CCR5 is required for its function as a receptor for diverse human and simian immunodeficiency virus envelope glycoproteins.Virology. 1998; 248: 357-371Crossref PubMed Scopus (67) Google Scholar), CXCR3 (IgG1, clone 1C6), and directly conjugated mouse anti-human CD45RA-FITC (IgG2b, clone HI100), CD69-PE (IgG1, clone FN50), CD3-FITC (IgG1, clone UCHT1), TCRαβ-FITC (IgM, clone T10B9.1A-31), and CD4-APC (IgG1, clone RPAT4) were obtained from PharMingen, Inc. (San Diego, CA). Recombinant human SDF-1α and TARC were purchased from Peprotech (Rocky Hill, NJ). ICAM-1 was purified from human tonsils as described.5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar Human jejunum, ileum, colon, lung, tonsil, and inflamed liver were obtained from patients undergoing various surgical procedures. Synovial fluid (SF) was obtained from patients undergoing diagnostic arthrocentesis. Peripheral blood was collected in heparinized tubes from healthy donors. Bronchoalveolar lavage (BAL) fluid was obtained by optic bronchoscopy from healthy volunteers. Skin lymphocytes were obtained from suction blisters in sensitized volunteers. All human subject protocols were approved by the Institutional Review Boards at Stanford University or Leicester University. Peripheral blood lymphocytes were isolated as described.5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar Briefly, after dextran sedimentation and separation of the mononuclear fraction over Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ), monocytes were removed by two 30 minute rounds of adherence to plastic culture flasks in an incubator. Tonsil lymphocytes were obtained by dispersal of fresh tonsils through a stainless steel mesh followed by incubation in a plastic flask to deplete adherent cells. Bronchoalveolar lavage subjects were premedicated with nebulized salbutamol, lightly sedated with midazolam and the upper airway was anesthetized with 2% lignocaine. 180 ml of normal saline were inserted through the bronchoscope into the middle lobe of the right lung and the lymphocyte-containing fluid was aspirated using gentle suction (fluid recovery was 20 to 46%). Lymphocytes from the epithelium and lamina propria of human intestine were isolated as described previously.16Fiocchi C Youngman K Isolation of human intestinal mucosal mononuclear cells.in: Collogan JE Kruisbeek AM Margulies DH Shevach EM Strober W Current Protocols in Immunology. John Wiley & Sons, Inc., New York1997: 7.30.1-7.30.8Google Scholar Briefly, the epithelium and any resident lymphocytes were removed from the lamina propria by gentle stirring in the presence of 1 mmol/L EDTA. The epithelium-free lamina propria was then dispersed through wire mesh. Lymphocytes were isolated from inflamed human skin as described previously.17Picker LJ Treer JR Ferguson-Darnell B Collins PA Bergstresser PR Terstappen LW Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-selective homing receptor for skin-homing T cells.J Immunol. 1993; 150: 1122-1136PubMed Google Scholar Briefly, skin lymphocytes were isolated from the fluid drained from suction blisters raised over the site of an epidermal delayed-type hypersensitivity reaction (to poison oak leaves or Candida albicans extract) elicited in allergic volunteers. Lymphocytes were isolated from normal lung, inflamed liver, and inflamed synovial fluid as described.4Kunkel EJ Campbell JJ Haraldsen G Pan J Boisvert J Roberts AI Ebert EC Vierra MA Goodman SC Genovese MC Wardlaw AJ Greenberg HB Parker CM Butcher EC Andrew DP Agace WW Lymphocyte CCR9 and epithelial TECK expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (548) Google Scholar Briefly, lung lymphocytes were obtained by mincing the tissue with fine scissors and passing the supernatant through gauze; liver lymphocytes were obtained after mechanical dispersal of liver pieces followed by Ficoll separation, and synovial fluid mononuclear cells were isolated by Ficoll separation. We performed confirmatory studies to investigate whether chemokine receptor expression on extracted lymphocytes might have been altered by the tissue-chelation procedure. In these parallel experiments, purified lymphocytes (from peripheral blood) were treated by chelation in exactly the same manner as the tissue samples. Donor-matched, untreated and treated lymphocytes were then stained with the same mAbs to chemokine receptors and adhesion molecules presented in the text. We found no staining differences between the treated and untreated cells (Kunkel and Campbell, unpublished findings). Chemotaxis assays were performed using 24-well Transwell plates (Corning Costar, Cambridge, MA; 5 μm pores) in RPMI 1640 supplemented with 0.5% bovine serum albumin for 2 hours in an incubator as described.5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar Migrated cells were stained with CD4-APC, CD45RA-CyChrome, CLA-FITC, and α4β7-PE to analyze T cell subsets (Pharmingen). Chemokine-triggered adhesion assays were performed as described.5Campbell JJ Haraldsen G Pan J Rottman J Qin S Ponath P Andrew DP Warnke R Ruffing N Kassam N Wu L Butcher EC The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells.Nature. 1999; 400: 776-780Crossref PubMed Scopus (739) Google Scholar Briefly, sorted lymphocytes were allowed to settle on glass slides coated with purified human ICAM-1 and stimulated to adhere with chemokines, after which the slides were gently washed and bound lymphocytes were fixed with gluteraldehyde and counted using NIH Image software (version 1.62b). Tissue or blood lymphocytes were stained and gated for the populations of interest using markers labeled with fluorescein isothyocyanate (FITC), phycoerythrim (PE), or allophycocyanin (APC). Unconjugated (or isotype-matched control mAbs) were detected using a biotinylated horse anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) and streptavidin-PerCP (Pharmingen). In some experiments, blood lymphocytes were stained for CCR4 and CD45RO and sorted on a FACSVantage SE (Becton-Dickinson). Four-color flow cytometry was carried out on a FACSCalibur (Becton-Dickinson) using CellQuest software, version 3.1 (Becton-Dickinson). In an attempt to more thoroughly understand the phenotype of tissue-infiltrating CD4+ lymphocytes, we have undertaken to gently isolate lymphocytes from surgical specimens for direct examination. Our lymphocyte isolation techniques involved chelation at 4°C as previously described.4Kunkel EJ Campbell JJ Haraldsen G Pan J Boisvert J Roberts AI Ebert EC Vierra MA Goodman SC Genovese MC Wardlaw AJ Greenberg HB Parker CM Butcher EC Andrew DP Agace WW Lymphocyte CCR9 and epithelial TECK expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (548) Google Scholar This technique allowed us to avoid prolonged (potentially destructive) exposure of lymphocytes to digestive enzymes at 37°C as entailed by conventional protocols. We first examined the activation and naive/memory status of tissue infiltrating cells. CD4+ lymphocytes from non-lymphoid tissues (eg, inflamed skin, normal jejunum, lung BAL fluid, and psoriatic arthritis SF in Figure 1, C–F) were overwhelmingly of the memory phenotype (CD45RA−). This is a dramatic enrichment of memory cells over their representation in blood, where they usually comprise less than 50% of CD4+ T cells (Figure 1A). In contrast, CD4+ T cells isolated from tonsil (a representative secondary lymphoid organ) maintained a naïve to memory ratio similar to that of blood (Figure 1B). Both lymphoid and non-lymphoid tissues contained large numbers of cells of activated phenotype (as assessed by the activation marker CD69, Figure 1). Activated cells were exceedingly rare in the blood (Figure 1A). To further characterize the tissue-derived lymphocytes, we examined their expression of the two best-studied adhesion molecules associated with tissue-specific homing: CLA and α4β7 integrin. CLA is a ligand for E-selectin, which allows CLA+ cells to interact with E-selectin in cutaneous venules. CLA+ cells home preferentially to cutaneous sites in vivo, and T cells specific for cutaneous antigens reside within this population.18Santamaria Babi LF Picker LJ Perez Soler MT Drzimalla K Flohr P Blaser K Hauser C Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen.J Exp Med. 1995; 181: 1935-1940Crossref PubMed Scopus (261) Google Scholar The α4β7 integrin is a ligand for MAdCAM-1, which allows α4β7+ cells to interact with MAdCAM-1+ intestinal venules. α4β7- memory cells home preferentially to intestinal sites in vivo,19Williams MB Butcher EC Homing of naive and memory T lymphocyte subsets to Peyer's patches, lymph nodes, and spleen.J Immunol. 1997; 159: 1746-1752PubMed Google Scholar and cells specific for intestinal antigens reside within this population.20Williams MB Rose JR Rott LS Franco MA Greenberg HB Butcher EC The memory B cell subset responsible for the secretory IgA response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, α4β7.J Immunol. 1998; 161: 4227-4235PubMed Google Scholar Memory CD4+ cells from blood contained a mixture of CLA-expressing, α4β7-expressing, and other less-well studied subsets (Figure 1, A; far right panel). However, when we examined cells isolated directly from skin or from jejunal lamina propria we found essentially pure CLA+ or α4β7+ populations, respectively (Figure 1, C and D; far right panels). In contrast, CD4+ populations from the lung or tonsil, where these molecules are proposed to play little or no role in homing, contained very few cells with high levels of these molecules (Figure 1, B and E; far right panels). Interestingly, lymphocytes from the synovial fluid of autoimmune arthritis patients appeared to be an exception to the rule of tissue-specific lymphocyte homing (eg, psoriatic arthritis in Figure 1E; far right panel). Although naïve cells were excluded from this extra-lymphoid site, the representation of memory cells expressing CLA or α4β7 was similar to that of blood. The above data suggested that the gentle method we used for isolating lymphocytes from tissues (see Materials and Methods)4Kunkel EJ Campbell JJ Haraldsen G Pan J Boisvert J Roberts AI Ebert EC Vierra MA Goodman SC Genovese MC Wardlaw AJ Greenberg HB Parker CM Butcher EC Andrew DP Agace WW Lymphocyte CCR9 and epithelial TECK expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity.J Exp Med. 2000; 192: 761-768Crossref PubMed Scopus (548) Google Scholar preserved the predicted homing phenotypes of the isolated cells. We then examined the expression of chemokine receptors by these cells, focusing on CCR4, CCR5, and CXCR3. The vast majority of CD4+ cells isolated from the 10 different non-lymphoid tissues examined (Figure 2) expressed CCR5 and CXCR3 (Figure 2, C -L; middle and right panels). This is in dramatic contrast to tonsil, where only very small numbers of CD4+ lymphocytes expressed CCR5 or CXCR3 (Figure 2B; far right panel). Nearly all of the CD4+ populations infiltrating non-lymphoid tissues contained far more CCR5+ and CXCR3+ cells than did memory CD4+ cells from the blood (with the exception of CXCR3 in the skin, where the pattern was similar to that on CLA+ cells in the blood11Qin S Rottman JB Myers P Kassam N Weinblatt M Loetscher M Koch AE Moser B Mackay CR The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions.J Clin Invest. 1998; 101: 746-754Crossref PubMed Scopus (1206) Google Scholar) (Figure 2A; middle and right panels). The ubiquity of CCR5+ and CXCR3+ T cells within so many diverse tissues suggests that CCR5 and CXCR3 are not involved in determining the tissue-specificity of lymphocyte homing, and that CCR5 and CXCR3 expression may be a general phenotype of tissue-infiltrating lymphocytes. CCR5 and CXCR3 may therefore be more important with respect to positioning or retention of lymphocytes after tissue entry.21Luster AD Chemokines: chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3275) Google Scholar Indeed, it is possible that these receptors may actually be induced after tissue entry. Unlike the ubiquitous expression pattern of CCR5 and CXCR3, lymphocyte CCR4 expression was variable among the tissues studied (Figure 2, C–L. CCR4 was not seen on lamina propria CD4+ cells from any of the intestinal segments (including jejunum, ileum and colon, Figure 2, D–F), or on liver-infiltrating CD4+ cells (Figure 2G). CCR4 was expressed at very high levels only in the skin (Figure 2C). CCR4 was expressed at lower levels by synovial fluid CD4+ cells from autoimmune arthritis patients (rheumatoid, Figure 2K and psoriatic, Figure 2L) but not from osteoarthritis patients (Figure 2J). CCR4 was also expressed at low levels on bronchial CD4+ cells (Figure 2I) and even more rarely by lung interstitial CD4+ cells (Figure 2H). Like CCR5 and CXCR3, there was little CCR4 expression in the tonsil (Figure 2B). The expression of CCR4 by skin-derived lymphocytes appeared significantly higher than its expression by bronchial or synovial cells. This higher expression was confirmed by additional experiments using blood CLA+ lymphocytes from tissue donors as an internal standard (Figure 3). Flow cytometry of CCR4 on
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