Search

Foreign body ingestion is a common reason for visiting the emergency room. Foreign bodies can lodge anywhere in the upper aerodigestive tract and can sometimes be difficult to extract. Wire bristles that dislodge from grill-cleaning wire brushes and which are then accidentally swallowed can be particularly challenging to remove due to their small size, propensity to become embedded, and their ability to migrate through tissues. This case reveals the speed with which wire bristle foreign bodies can migrate through tissues and exemplifies the need to obtain computerized tomography evaluations in close proximity to any planned attempt to remove them.

Aim: To examine the pre- and post-impact activation of five upper extremity muscles in the tennis volley across conditions of ball speed, ball type and side of the body.Methods: A repeated measures design in a biomechanics laboratory setting was used. A total of 24 recreational tennis players (mean (SD) age 24 (5) years, height 176 (10) cm, mass 76 (13) kg) were recruited from a university. Participants performed tennis volleys under 18 ball conditions: three ball speeds (slow, medium and fast), with three ball types (two oversize and one regular size) each from two sides (forehand and backhand). Average normalised electromyographic levels of the flexor carpi radialis, extensor carpi radialis, triceps brachii, anterior/middle deltoid and posterior/middle deltoid of the hitting arm during pre- and post-impact phases (200 ms before and after ball-racquet impact, respectively) were assessed.Results: For the pre-impact phase, a significant muscle and side interaction (p<0.001) and significant main effects for speed (p = 0.002) and muscle (p<0.001) were observed. For the post-impact phase, significant interactions were observed for ball type and side (p = 0.002), ball speed and side (p = 0.011) and muscle and side (p = 0.001), as well significant main effects for muscle (p<0.001), speed (p = 0.035) and side (p<0.001).Conclusion: Oversize tennis balls do not significantly increase upper extremity muscle activation compared to regular size balls during a tennis volley. The highest post-impact activation was observed in the ECR indicating a vigorous wrist stabilisation role that could irritate players with lateral epicondylalgia. [ABSTRACT FROM AUTHOR]

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Recently, Thrasher et al. (College Student Affairs Journal 27(1): 57-75, 2007) explored the efficacy of the Theory of Reasoned Action (TRA; Ajzen and Fishbein, Attitudes, personality, and behavior, 1980) in explaining gambling behavior of college students. However, their study found the TRA only predicted small amounts of variance in gambling intentions. Heeding their call to enhance the efficacy of the TRA through the addition of explanatory variables to the model, the present study incorporated gambling motivations and locus of control as moderating variables within the TRA to test the potential of a modified TRA in explaining gambling behavior of college students. A total of 345 students at a major metropolitan research university in the Midwest volunteered to participate in the study. A series of hierarchical linear regressions indicated intrinsic motivation to accomplish (p = .002) significantly moderated the relationship between gambling attitudes and gambling intentions. Further, internal locus of control (p < .001), chance locus of control (p < .001), and powerful others locus of control (p < .001) also significantly moderated the relationship between gambling attitudes and gambling intentions. The significant impact of the moderating variables on the relationship between gambling attitudes and intentions suggests intrinsic motivation and locus of control can alter the impact of the relationship between gambling attitudes and gambling intentions.

High-throughput screening of the corporate compound collection led to the discovery of a novel series of N-substituted-5-aryl-oxazolidinones as potent human CCR8 antagonists. The synthesis, structure-activity relationships, and optimization of the series that led to the identification of SB-649701 (1a), are described.

CCL1, the CCR8 ligand, is a CC chemokine secreted by activated monocytes and lymphocytes and is a potent chemoattractant for these cell types. The in vivo role of the CCL1/CCR8 axis in Th2-mediated inflammation is far from clear. Ligand neutralisation studies reported discrepancies in the effect of CCL1/CCR8 and CCR8 knockout studies showed very different insights into the functional role of the CCR8. To further study the biological function of CCL1, we focused on the generation and characterisation of RNA aptamers. We report here the in vitro isolation of the first nuclease resistant and selective RNA aptamer (T48) with high-binding affinity for human and mouse CCL1. The T48 aptamer but not a random control aptamer antagonises CCL1 function in a dose-dependent fashion in both heparin binding and chemotaxis assays. To our knowledge, the T48 aptamer constitutes one of the most potent CCL1 antagonists reported to date and is an excellent tool to dissect CCL1-specific function in vivo. The T48 aptamer may also have potential as new generation of therapeutic tools.

CXCL10 (also known as IP-10 in humans and CRG-2 in mice) is a nonglycosylated chemokine and a member of the non-ELR CXC chemokine subfamily implicated in a variety of inflammatory conditions. The role of CXCL10 in different disease states still requires clarification, and new approaches are necessary to better understand its biological function. We report here the isolation of a series of nuclease-resistant RNA aptamers that act to antagonize human CXCL10 function in a number of in vitro and cell-based assays. The two most potent aptamers identified were highly selective for human CXCL10. A further aptamer was identified that antagonized both the human and the mouse CXCL10. A combination of a molecular-biology-based truncation and solid-phase synthesis enabled the truncation of one of the aptamers from 71 to 34 nucleotides. This was followed by PEGylation, 3' capping, and further stabilization of the RNA aptamer, while its high potency was maintained. These aptamers could be utilized as powerful target validation tools and may also have therapeutic potential. To our knowledge, the CXCL10 aptamers generated are the most potent antagonists of CXCL10/CXCR3 signaling reported to date.

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.

The purpose of this study was to examine the effect of ball size on reaction time (from ball projection to initial racket movement), movement time (from ball projection to ball-racket impact), mean rectified acceleration and vibration frequency of the racket during the impact phase (100 ms duration after ball-racket impact) of the tennis volley. Twenty-nine beginning to intermediate level tennis players performed volleys under 18 experimental conditions including variations in lateral contact location (forehand and backhand), ball type (Penn oversize, Wilson oversize, regular size), and ball speed (fast, medium, slow). A ball machine was shielded so that the subjects could not predict the ball trajectory before it was released from the machine. Outcome measures were determined using a miniature uni-axial accelerometer and a Photogate timing device. ANOVA with repeated measures and Tukey's post hoc tests were used in statistical analyses (p < .025). The reaction times for the fast speed were significantly shorter than the corresponding times for the slow speed and the reaction times for the forehand volley were significantly shorter than that of the backhand volley. The significant interaction in movement time between ball speed and ball type indicates that the benefits of the larger ball (to slow the game down) increase as the ball speed increases. No significant effect of ball type or ball speed, or interactions were found on mean rectified acceleration and vibration frequency. These results suggest that oversized balls would not cause an increased load to the hitting arm while executing a tennis volley.

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.

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 gamma. 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 approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% 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.

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.

Background & Aims: Thymus-expressed chemokine (TECK) or CCL25) is selectively expressed in the small bowel (SB), where lamina propria lymphocytes (LPL) and intraepithelial leukocyte expressing the cognate chemokine receptor CCR9 predominate. We characterize the role of TECK and CCR9-expresing lymphocytes in small intestinal Crohn's disease., Methods: CCR9 expression on lymphocytes from lamina propria, mesenteric lymph node, and peripheral blood was analyzed by flow cytometry and by Northern blotting for LPL. TECK expression was analyzed in inflamed SB and colon by reverse-transcription polymerase chain reaction and immunohistochemistry., Results: The fraction of CCR9(+) T cells in inflamed SB was significantly lower than in uninvolved SB mucosa. In contrast, in peripheral blood lymphocytes, CCR9(+) lymphocytes were markedly elevated in patients with small bowel Crohn's or celiac disease, but not in patients with purely colonic Crohn's. Also, TECK expression is altered in inflamed small bowel, being intensely expressed in a patchy distribution in crypt epithelial cells in proximity to lymphocytic infiltrates. TECK is not expressed in either normal or inflamed colon., Conclusions: In SB immune-mediated diseases, there is repartitioning of CCR9(+) lymphocytes between SB and blood and an altered pattern of TECK expression in SB Crohn's. The TECK/CCR9 ligand/receptor pair may play an important role in the pathogenesis of SB Crohn's disease.

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 alpha (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.

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 alpha(4)beta(7). 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, 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 (alpha(4)beta(7)(-)) 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-gamma 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.

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 Ckbeta-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 alpha(4)beta(7). 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.

The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

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 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 (alpha4beta7+) 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.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31), a member of the Ig superfamily, is expressed strongly at endothelial cell-cell junctions, on platelets, and on most leukocytes. CD31 has been postulated to play a role in vasculogenesis and angiogenesis, and has been implicated as a key mediator of the transendothelial migration of leukocytes. To further define the physiologic role of CD31, we used targeted gene disruption of the CD31 gene in embryonic stem cells to generate CD31-deficient mice. CD31-deficient mice (CD31KO) are viable and born at the expected Mendelian frequency, remain healthy, and exhibit no obvious vascular developmental defects. In response to inflammatory challenge, polymorphonuclear leukocytes of CD31KO mice are arrested between the vascular endothelium and the basement membrane of inflammatory site mesenteric microvessels, confirming a role for CD31 in the migration of neutrophils through the subendothelial extracellular matrix. Normal numbers of leukocytes are recovered from inflammatory sites in CD31KO mice, however, suggesting that the defect in leukocyte migration across basal lamina observed in the absence of CD31 may be compensated for by the use of other adhesion molecules, or possibly an increased rate of migration. Homing of T lymphocytes in vivo is normal, and CD31KO mice are able to mount a cutaneous hypersensitivity response normally. In addition, CD31-mediated homophilic adhesion does not appear to play a role in platelet aggregation in vitro. This study provides genetic evidence that CD31 is involved in transbasement membrane migration, but does not play an obligatory role in either vascular development or leukocyte migration.

STCP-1 stimulated T cell chemoattractant protein-1 (STCP-1) (macrophage-derived chemokine; MDC), a recently described CC chemokine for chronically activated T lymphocytes, was found to act specifically on a subset of memory CD4 lymphocytes that displayed a Th2 cytokine profile. Also, STCP-1, thymus and activation regulated chemokine (TARC), eotaxin, and eotaxin-2 acted specifically on in vitro derived Th2 lymphocytes, while IP-10 (IFN-gamma-inducible 10-kDa protein) showed some preference for Th1 lymphocytes. The corresponding receptors for eotaxin, TARC, and IP-10 are also differentially expressed on Th1 and Th2 lymphocytes. In desensitization Ca flux experiments, TARC and STCP-1 bound to a common receptor and therefore at least one chemokine receptor for STCP-1 is CCR4. STCP-1 expression is restricted to immune cells. Dendritic cells, B cells, and macrophages produce STCP-1 constitutively, while NK cells, monocytes, and CD4 lymphocytes produce STCP-1 upon appropriate stimulation. Production of STCP-1 is positively modulated by Th2 cytokines IL-4 and IL-13 but inhibited by IL-10.

CD31 or platelet/endothelial cell adhesion molecule (PECAM-1) is a 130-kDa glycoprotein expressed on endothelial cells, granulocytes, a subset of lymphocytes and platelets. In this study, we examined the ability of four monoclonal antibodies (mAb) against different domains of CD31 to modulate the function of T lymphocytes, monocytes and neutrophils. Engagement of CD31 on T lymphocytes results in co-stimulation of T lymphocyte proliferation to suboptimal doses of anti-CD31 mAb. This proliferation is accompanied by secretion of numerous cytokines and chemokines, up-regulation of CD25 and an increase in cell size. Purification of T lymphocytes into CD45RO and CD45RA subsets showed that only naive CD45RA T lymphocytes are co-stimulated by anti-CD31 mAb. Further studies on neutrophils show that engagement of CD31 results in down-regulation of CD62L and up-regulation of CD11b/CD18 as well as oxidative burst, as assessed by superoxide release. In addition, ligation of CD31 on monocytes results in TNF-alpha secretion, and studies with various cell signaling inhibitors indicate that tyrosine kinases and cAMP-dependent kinases are involved in monocyte activation via CD31. Of the four mAb used in this study, only two activated human leukocytes. These mAb were PECAM-1.3 and hec7, which bind to domains 1 and 2 of CD31. We conclude that engagement of domains 1 and 2 of CD31 results in outside-in signaling in leukocytes.

The leukocyte integrin LFA-1 plays an important role in leukocyte trafficking and the immune response. Using LFA-1-deficient mice, we demonstrate that LFA-1 regulates the trafficking of lymphocytes to peripheral lymph nodes, and, to a lesser degree, to mesenteric lymph nodes and acute inflammatory sites. LFA-1, either because of its role in initial adhesion and/ or the passage of leukocytes across endothelial cells, plays a vital role in T lymphocyte and neutrophil transendothelial migration. Neutrophils and activated T lymphocytes from LFA-1-deficient mice were unable to cross endothelial cell monolayers in response to a chemokine gradient, whereas wild-type (WT) T lymphocytes and neutrophils were capable of migration. By contrast, LFA-1-deficient T lymphocytes displayed normal chemotaxis to the same chemokine. Our studies with LFA-1-deficient monocytes indicate that LFA-1 acts in concert with complement receptor 3 to mediate transendothelial migration of these cells, as anti-CD18 monoclonal antibodies (mAb) blocked both WT and LFA-1-deficient monocyte transendothelial migration, whereas anti-CD11 b mAb preferentially blocked transendothelial migration of LFA-1-deficient monocytes. Finally, whereas anti-CD31 mAb blocked WT monocyte and neutrophil transendothelial cell migration they did not block LFA-1-deficient monocyte and neutrophil transendothelial migration.

Carbohydrate ligands for lymphocyte L-selectin are expressed on high endothelial venules (HEVs) in peripheral lymph nodes and sites of chronic inflammation and mediate the recruitment of lymphocytes from the blood into these tissues. In the mouse, these ligands, collectively termed the peripheral lymph node addressin (PNAd), have been shown to contain fucose, sialic acid, and sulfate and to include several HEV glycoproteins including GlyCAM-1, CD34, and MAdCAM-1. Monoclonal antibody (MAb) MECA-79, which binds a sulfate-dependent epitope, recognizes PNAd in both mouse and man. In humans, only CD34 has been identified among the glycoprotein species that react with MECA-79. Although P-selectin is highly expressed in tonsil HEVs, it was not found to react with MECA-79 or to support L-selectin-mediated lymphocyte rolling. To further characterize human PNAd, MAbs were developed against purified PNAd immunoisolated from human tonsil. MAbs JG-1, JG-5, JG-9, and JG-10, like MECA-79, bind HEVs in human tonsil and react similarly in Western blots, and JG-9 and JG-10 also block lymphocyte rolling on purified PNAd. In addition, by competitive ELISA on purified tonsil PNAd, all MAbs were found to react with overlapping epitopes. However, JG-1, JG-5, JG-9, and JG-10 do not recognize mouse PNAd, and unlike MECA-79, they recognize determinants that are sensitive to neuraminidase. Strikingly, the epitope recognized by JG-1, although abundant in tonsil and peripheral lymph node, is absent from appendix HEVs or HEVs in some samples of chronically inflamed skin, even though these HEVs are MECA-79 reactive. Moreover, although JG-5 and JG-9 react well with tonsil, peripheral lymph node, and inflamed skin HEVs, they react only with occasional endothelial cells in appendix tissues. These findings point to significant diversity in the carbohydrate determinants expressed by HEVs and recognized by L-selectin and demonstrate their differential representation in different sites in vivo. These antibodies should be useful in probing the precise structure of human L-selectin ligands.

A novel human chemokine STCP-1 (stimulated T cell chemotactic protein) was isolated from an activated macrophage cDNA library. The chemokine has four cysteines positioned in a manner that identifies STCP-1 as a member of the CC chemokine family. The amino acid sequence shows 34% identity with RANTES. The gene consists of 3 exons and 2 introns with the position of intron/exon boundaries similar to that of RANTES. The gene is expressed as a 3.4-kilobase transcript on lymph node, thymus, and Appendix. STCP-1 induces Ca2+ mobilization in a small percentage of primary activated T lymphocytes, but on repeated stimulation the percentage of T lymphocytes that respond to STCP-1 increases. The chemokine STCP-1 does not induce Ca2+ mobilization in monocytes, dendritic cells, neutrophils, eosinophils, lipopolysaccharide-activated B lymphocytes, and freshly isolated resting T lymphocytes. Similarly, STCP-1, while acting as a mild chemoattractant for primary activated T lymphocytes, is a potent chemoattractant for chronically activated T lymphocytes but has no chemoattractant activity for monocytes, neutrophils, eosinophils, and resting T lymphocytes. As STCP-1 acts specifically on activated T lymphocytes, it may play a role in the trafficking of activated/effector T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology.

Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

T cells show a bias in their migration pathways: some T cells preferentially migrate to peripheral lymph nodes (LN), some to mucosal tissues, and some to peripheral tissues such as skin. These recirculation pathways were examined in sheep by collecting lymph draining into and out of peripheral and intestinal LN, and using fluorescent dyes to trace the recirculation of the lymph cells. Monoclonal antibodies (mAb) to alpha 4, beta 1, and beta 7 integrins, and L-selectin, were used to define three major populations of recirculating T cells. Naive-type T cells (L-selectin+, alpha 4 beta 1lo beta 7lo) migrated preferentially through peripheral LN. Two memory populations could be defined: alpha 4 beta 1hi beta 7- and alpha 4 beta 7hi beta 1lo. alpha 4 beta 1hi beta 7- T cells were present in lymph draining from the skin. T cells migrating preferentially through intestinal LN were alpha 4 beta 7hi beta 1lo. Consistent with this migration pattern, the endothelial receptor for alpha 4 beta 7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was detected on high endothelial venules within intestinal LN and Peyer's patches, but only weakly on high endothelial venules within peripheral LN. Thus, there are at least three easily definable subsets of T cells, based on integrin expression, which show distinct migration preferences.

The leukocyte integrin alpha 4 beta 7 is a receptor for the vascular mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Most circulating B and T lymphocytes in man are alpha 4+, and on these cells the regulated display of the beta 7 integrin chain determines the expression of alpha 4 beta 7 and, in large part, binding to MAdCAM-1. Among CD4+ T cells, beta 7 high memory cells (including the L-selectin+ subset) bind MAdCAM-1 better than beta 7int naive cells; whereas beta 7- memory cells,including skin homing lymphocytes, interact poorly if at all. Circulating alpha E beta 7+ T cells are alpha 4 beta 7high and also bind MAdCAM-1 well. B cells are also subdivided by beta 7 expression, and beta 7+ B cells bind MAdCAM-1 better than the beta 7low/- subset. The related vascular ligand vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelium primarily in nonmucosal sites of inflammation, interacts with blood lymphocytes (including beta 7high T cells) almost exclusively via alpha 4 beta 1 and binds beta 7low/-(beta 1high) better than beta 7+ B cells and memory cells better than naive CD4+ cells. beta 7-(beta 1high) memory T cells are somewhat enriched over beta 7high memory cells at low (but not at high) VCAM-1 densities. Interestingly, CD56+ NK cells, which express both alpha 4 beta 7 and alpha 4 beta 1, bind well to VCAM-1 but poorly to MAdCAM-1. The findings indicate that the display and function of alpha 4 beta 7 determine integrin-dependent blood lymphocyte interactions with MAdCAM-1, thus delineating discrete mucosal vs nonmucosal lymphocyte populations in vivo; that alpha 4 beta 1 dominates blood lymphocyte interactions with VCAM-1; and that quantitative and qualitative regulation of MAdCAM-1 vs VCAM-1 can critically control the recruitment of specialized lymphocyte subsets during inflammation.

Beta 7 is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of beta 7. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express alpha 4 beta 7 and alpha E beta 7 using mAb against alpha E, beta 7 and mAb DATK32 which recognizes a combinatiorial epitope on alpha 4 beta 7+ thymocytes have a mature phenotype: TcR+, CD11a(hi)CD44(hi)HSA(dull). Small subsets of double-negative CD4-CD8-, single-positive CD4+ and CD8+ thymocytes express beta 7, while double-positive CD4+CD8+ thymocytes are beta 7-. However, two integrins alpha E beta 7 and alpha 4 beta 7 recognized by anti-beta 7 are not expressed on an identical subpopulation of thymocytes, as alpha E beta 7+ alpha 4 beta 7-, alpha E beta 7 + alpha 4 beta 7+ and alpha E beta 7- alpha 4 beta 7+ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of alpha E beta 7 but little alpha 4 beta 7. In the spleen, Peyer's patches and lymph nodes, alpha 4 beta 7 is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4+ and CD8+ lymphocytes, express high levels of beta 7 in the form of alpha 4 beta 7 and alpha E beta 7, although, as observed with lymphocytes, not all alpha 4 beta 7 hi CD4+ lymphocytes expressed alpha 4 beta 7. The population of alpha 4 beta 7 hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4+ lymphocyte population, which can be further subdivided on the basis of alpha E beta 7, L-selectin and alpha 4 expression. Therefore, memory CD4+ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.

We describe here a mAb, DATK44, which induces homotypic aggregation of TK1 cells (a CD8 lymphoma). The glycoprotein recognized by DATK44 is of approximate m.w. 50 kDa and is expressed by monocytes, neutrophils, and subsets of lymphocytes, as well as on the high endothelial venule in peripheral and mesenteric lymph nodes. We named this Ag TABS (T cell activation B cell subset Ag), as TABS appears on T lymphocyte activation and is expressed at low and high levels by B cells. TABS is differentially regulated during T lymphocyte development, CD4+veCD8+ve thymocytes being TABShigh, while single positive CD4+ve and CD8+ve thymocytes are TABSdull CD4-veCD8-ve thymocytes are clearly split into dull and bright populations by the mAb. On exit from the thymus, T lymphocytes cease to express TABS, but T lymphocyte activation results in re-expression of TABS. TABS also shows tight coregulation with heat stable Ag on resting lymphocytes, but coexpression of these two molecules is lost upon lymphocyte activation. DATK44-induced aggregation of TK1 cells is temperature sensitive and blocked by pretreatment of the cells with metabolic inhibitors, genestein, dibutyl cAMP or cytochalasin B, while colchicine, staurosporin, sphingosine, okadaic acid, and W7 are without effect. DATK44-induced TK1 cell aggregation appears to be mediated by the LFA-1 pathway, as aggregation is blocked by anti-LFA-1 and anti-ICAM-1 mAbs but not by Abs capable of blocking CD44 and alpha 4 beta 7-mediated adhesion. Thus, TABS appears to be an adhesion inducer that selectively activates LFA-1-mediated lymphocyte aggregation.

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

Lymphocytes continuously migrate throughout the body in search of antigens. Virgin lymphocytes recirculate freely between the blood and different lymphatic organs, whereas immunoblasts extravasate preferentially into sites similar to those where they initially responded to antigen. Tissue-specific extravasation of lymphocytes is largely controlled by distinct lymphocyte surface receptors that mediate lymphocyte binding to high endothelial venules (HEV). In the present study, the molecular mechanisms determining the specificity of human mucosal (lamina propria) lymphocyte binding to different endothelial recognition systems were analyzed. Mucosal immunoblasts adhered five times better than small mucosal lymphocytes to mucosal HEV. Importantly, mucosal immunoblasts also bound to synovial HEV almost as efficiently as to mucosal HEV, but they did not adhere to peripheral lymph node HEV. To study the impact of different homing-associated molecules in this dual endothelial binding, we used a gut-derived T cell line and freshly isolated mucosal immunoblasts. Both cell types expressed integrins alpha 4, beta 1, beta 7, and lymphocyte function associated antigen 1 (LFA-1), and were CD44 positive, but practically L-selectin negative. Binding of mucosal immunoblasts to mucosal HEV was almost completely abolished by pretreatment with anti-beta 7 monoclonal antibodies, but it was independent of alpha 4/beta 1 function. In contrast, alpha 4/beta 1 partially mediated immunoblast adherence to synovial HEV, whereas alpha 4/beta 7 had only a minor role in adherence of blasts at this site. CD44 and LFA-1 contributed to HEV-binding both in mucosa and synovium. Taken together, this is the first report that demonstrates a critical role for alpha 4/beta 7 in the binding of gut lymphocytes to mucosal venules in humans. Moreover, a hitherto unknown interaction between mucosal effector cells and synovial endothelial cells was shown to be only partially mediated by the currently known homing receptors. The dual endothelial binding capacity of mucosal blasts may help to explain the pathogenesis of reactive arthritis not uncommonly associated with inflammatory and infectious bowel disease.

The mouse CD8+ T cell lymphoma TK1 expresses high levels of alpha 4 beta 7 integrin, which it can use to interact with multiple ligands including mucosal addressin-1 (MAdCAM-1), VCAM-1, and fibronectin. In addition, alpha 4 beta 7 can support TK1 cell aggregation. Here we have produced and characterized a panel of mAbs against alpha 4 beta 7 to define antigenic and functional epitopes associated with its distinct functions. One mAb, DATK32, is unique in recognizing an epitope specific to the alpha 4 beta 7 heterodimer. Furthermore, DATK32 induces TK1 cell aggregation yet inhibits TK1 cell adhesion to MAdCAM-1, VCAM-1, and fibronectin. Considered as a whole, the panel of anti-alpha 4 beta 7 mAbs studied define unique patterns of inhibition for alpha 4 beta 7 binding to each of its defined molecular ligands. We conclude that alpha 4 beta 7 interactions with MAdCAM-1, VCAM-1, and fibronectin can be modulated by Ab binding to distinct epitopes and thus probably involve functionally separable, although physically overlapping binding sites on this multifunctional integrin. These findings are consistent with the general observation that integrins use distinct, potentially differentially regulated interaction sites for adhesion to multiple ligands. Extension of these concepts to alpha 4 beta 7 has important considerations for understanding the roles of this integrin in lymphocyte homing to mucosal sites and in cell-cell interactions during the immune response.

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

Lymphocyte recirculation through different organs is thought to be regulated by adhesion molecules ("homing receptors") recognizing tissue-specific vascular addressins on endothelium. Here we show that the alpha 4/beta 7-integrin has a key role in the migration of mouse lymphocytes to mucosal sites. Homing to Peyer's patches but not to peripheral lymph nodes is inhibited by Fab fragments of mAb PS/2 against the alpha 4-integrin chain, by mAb DATK32 recognizing a combinatorial epitope on the alpha 4/beta 7-integrin, and by mAb FIB30 against the beta 7-chain. The Abs significantly reduce homing of lymphocytes to the intestine, as well. The migration of immunoblasts to gut and gut-associated lymphoid tissue also involves the alpha 4/beta 7-integrin heterodimer. Another anti-alpha 4 Ab, R1-2, which blocks lymphocyte binding to Peyer's patches in the Stamper-Woodruff frozen section assay and lymphocyte adhesion to VCAM-1 and fibronectin, has only minor effects on lymphocyte traffic in vivo. Anti-VCAM-1 Ab as well as the fibronectin peptide CS-1 are without influence on the migration to Peyer's patches or intestine, in contrast to Ab against the mucosal addressin MAdCAM-1. Thus, homing to gut-associated sites is regulated by the alpha 4/beta 7-integrin heterodimer interacting with the vascular addressin, MAdCAM-1, and not with fibronectin or VCAM-1 as counterstructures. Inhibition of homing to Peyer's patches and intestine by the anti-integrin Abs studied was only partial. L-selectin also participates in the homing of small lymphocytes to mucosal sites, especially Peyer's patches, but does not contribute substantially to the localization of blasts into the intestinal wall. The results support a major, but not exclusive role of the alpha 4/beta 7-integrin in lymphocyte traffic to mucosal sites.

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 Peyer's 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.

1. The modification of the vasodilator effect of calcitonin gene-related peptide (CGRP) by a panel of monoclonal antibodies (MAbs), which map to discrete epitopes on the CGRP molecule, was investigated in pig coronary artery rings (PCA). The preparations were pre-constricted with acetylcholine (3 x 10(-7) M) and concentration-response curves to CGRP (2 x 10(-10)-2.56 x 10(-8) M) were obtained in the presence or absence of each MAb. 2. CGRP caused a concentration-dependent relaxation of PCAs which reached a maximum (98.2 +/- 4.8%, n = 25) at 1.28 x 10(-8) M and gave an EC50 of 3.8 +/- 0.8 x 10(-9) M. 3. Two MAbs which map to the N-terminal, CN1 and CRA3, did not affect the CGRP response whilst a third, CRA5, significantly inhibited its effect. 4. The C-terminal MAb, CRA2, did not modify the CGRP response whilst, in contrast, CB3 (C-terminal) potentiated its effect. A similar augmentation of the CGRP-induced vasodilatation was seen in the presence of the middle-region MAb, CRA8. 5. These results suggest that regional specific MAbs can modify the vasodilator effect of CGRP causing either inhibition (CRA5, N-terminal) or potentiation (CB3, C-terminal; CRA8, middle region).

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.

A panel of 18 monoclonal antibodies was raised to the human calcitonin gene related peptide (CGRP). Of these mabs, seven were specific for alpha CGRP and five for beta CGRP, while the remainder reacted with both alpha and beta CGRP. Nine different epitopes on CGRP were defined with these mabs. In addition, the mabs were tested in various combinations to develop a series of two site assays specific for alpha or for beta CGRP as well as assays able to detect both.