Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4+ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4+ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4+ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-gamma and TNF-alpha but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-gamma- and TNF-alpha-producing effector CD4+ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4+ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4+ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-Lhi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-Lhi CD4+ T cells were enriched in IFN-gamma-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-Llo CD4+ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4+ T blasts that were almost exclusively P-Lhi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4+ T cells residing in inflamed and non-inflamed tissues during the development of colitis.     

Selective requirements for leukocyte adhesion molecules in models of acute and chronic cutaneous inflammation: participation of E- and P- but not L-selectin

Adhesion molecules borne by both endothelial cells and circulating leukocytes are in large measure responsible for guiding the process of extravasation. The selectin family has been primarily associated with the early stages of adhesion involving initial contact and rolling. A significant body of evidence has accumulated indicating a fundamental role for the endothelial members of this family, E- and P-selectin, in a variety of inflammatory states and models. Although originally identified as the lymph node-specific lymphocyte homing receptor, L-selectin has also been suggested to play an important role in leukocyte recruitment to sites of inflammation. We have recently demonstrated, using L-selectin-deficient mice, that defects in contact hypersensitivity (CHS) responses are in essence due to the inability of T cells to home to and be sensitized within peripheral lymph nodes, whereas nonspecific effector cells are fully capable of entry into sites of cutaneous inflammation (Catalina et al, J Exp Med 184:2341, 1996). In the present study, we perform an analysis of adhesion molecule usage in two models of skin inflammation and show in both L-selectin-deficient as well as wild-type mice that a combination of P- and E-selectin is crucial for the development of both acute (croton oil) and chronic (contact hypersensitivity) inflammation at sites of the skin, whereas L-selectin does not appear to play a significant role. Moreover, alpha4 integrins are shown to be integral to a CHS but not an acute irritant response, whereas CD44 does not significantly contribute to either. These results provide a systematic examination in one study of major adhesion molecules that are critical in acute and chronic skin inflammation. They reinforce the essential role of the collaboration of E- and P-selectin in both specific and nonspecific skin inflammatory responses and the importance of alpha4 in the specific response only. In addition, they substantiate only a limited role, if any, for L-selectin in these cutaneous effector mechanisms and demonstrate the essential equivalence in this analysis of L-selectin-deficient mice compared with normal mice treated with blocking antibodies.     

Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation

The phenotype of T cells in the central nervous system (CNS) in two models of chronic inflammation (experimental allergic encephalomyelitis and Corynebacterium parvum-induced inflammation) was compared to that of T cells in gut and chronically inflamed subcutaneous tissue and lung. CNS T cells display a similar phenotype in both inflammatory models, and are phenotypically unique compared to T cells from the other inflamed tissues. T cells from inflamed CNS are mainly CD4+ and are the only population examined that express a typical activated/memory phenotype: CD44high/LFA-1high/ICAM-1high/CD45RBlow. The CNS T cells are alpha4beta7-integrin(negative), but express alpha4-integrin and activated beta1 integrin, suggesting expression of the alpha4beta1-heterodimer in an activated state. In contrast, most T cells in gut express low levels of activated beta1 integrin. The CNS T cells lack expression of alpha6 and alphaE integrin chains and L-selectin. In inflamed CNS and inflamed subcutaneous tissue, approximately 50% of T cells express high affinity ligands for P-selectin while fewer than 10% express high affinity ligands for E-selectin. In summary, our data show that, independent of the inflammatory stimulus, T cells recruited into the inflamed CNS are phenotypically distinct from T cells in other inflamed tissues. This finding leads us to hypothesize the existence of a phenotypically distinct 'CNS-seeking' T lymphocyte population.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression

The alpha1,3-fucosyltransferase, FucT-VII, is crucial for the formation of ligands for all three selectins, and its expression regulates the synthesis of these ligands. Short-term polarized T helper (Th)1, but not Th2 or naive CD4(+) T cells, can home to sites of inflammation, but the molecular basis for this difference has remained unclear. Here we show that naive CD4(+) T cells do not express FucT-VII and fail to bind vascular selectins. We also show that when CD4(+) T cells are activated in the presence of the Th1 polarizing cytokine interleukin (IL)-12, levels of FucT-VII mRNA and binding to E- and P-selectin are significantly augmented. In contrast, activation of CD4(+) T cells in the presence of IL-4, a Th2 polarizing cytokine, inhibited FucT-VII expression and binding to vascular selectins. T cell activation upregulated expression of the Core2 transferase, C2GnT, equivalently regardless of the presence or absence of polarizing cytokines. These data indicate that the selective ability of Th1 cells, as opposed to Th2 cells or naive CD4(+) T cells, to recognize vascular selectins and home to sites of inflammation is controlled principally by the expression of a single gene, FucT-VII.     

L-selectin from human, but not from mouse neutrophils binds directly to E-selectin

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.     

The importance of L-arginine metabolism in melanoma: an hypothesis for the role of nitric oxide and polyamines in tumor angiogenesis

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.     

Adhesion molecules in atopic dermatitis: patch tests elicited by house dust mite

Different T-helper subsets, which are characterized by the secretion of distinct cytokines (Th1, Th2), have been found in house dust mite-exposed skin of sensitized individuals and in nickel-specific T lymphocytes from nickel contact allergic and non-allergic individuals. In order to evaluate the role which adhesion molecules may play in the homing of different T-cell subsets into allergen-exposed skin of atopic and normal individuals, we compared the expression pattern of adhesion molecules in patch test reactions to house dust mite antigen (D.pt.), nickel sulfate (Ni) and the irritant anthralin. Biopsies were taken at various time points after application of these agents and studied by immuncytochemistry. To exclude an endogenous difference in adhesion molecule expression in atopic and non-atopic skin, sequential biopsies from Ni patch tests of 2 normal individuals were also included in this study. The expression of E-selectin, P-selectin, CD31, VCAM-1 and ICAM-1 on endothelial cells and other cells in the skin was quantified by microscopic evaluation. Skin homing T cells were also quantified using antibodies to CD3, CD4, CD8, UCHL-1, L-selectin and the cutaneous lymphocyte antigen (CLA). Independent of the eliciting substance, all lesions showed an upregulation of all adhesion molecules tested, with the exception of CD62. The appearance of E-selectin and an increase in ICAM-1 and VCAM-1 expression were first observed at 12 h after application of the various agents. In parallel, the number of CLA+ and L-selectin+ lymphocytes increased steadily. No principle differences could be established between the various types of skin reactions in atopic individuals, nor did the skin of patients with AD differ from normal controls. Our results provide evidence that differential expression of adhesion molecules does not play a major part in observed differential homing of Th1 and Th2-cell subsets into patch test sites provoked by house dust mite and nickel sulfate in atopic and non-atopic individuals.     

Contribution of endothelial selectins and alpha 4 integrins to eosinophil trafficking in allergic and nonallergic inflammatory reactions in skin

The role of endothelial selectins in mediating eosinophil recruitment was assessed using the trafficking of 111In-labeled blood eosinophils in mouse skin. An intradermal injection of chemoattractants (leukotriene B4, macrophage inflammatory protein-l alpha, and eotaxin) resulted in a rapid accumulation of 111In eosinophils that was reduced 49 to 91% by anti-P-selectin mAb. An anti-E-selectin mAb was ineffective, although a combined E- and P-selectin blockade resulted in >95% inhibition of all responses. The accumulation of a pulse of 111In eosinophils at sites of active cutaneous anaphylaxis (ACA) at 4 to 8 h and at 20 to 24 h after Ag challenge was completely dependent upon E- and P-selectin in combination, but not in isolation. In contrast, at 20 to 24 h after Ag challenge in a delayed-type hypersensitivity (DTH) reaction in skin, 111In eosinophil accumulation was largely independent of endothelial selectins, even when L-selectin was also blocked. An anti-alpha 4 integrin mAb significantly reduced 111In eosinophil trafficking in both allergic reactions but was slightly more effective in the DTH reaction compared with the ACA reaction. These results show that P-selectin and to a lesser extent E-selectin mediate eosinophil recruitment in skin in acute inflammatory reactions. In allergic, late-onset inflammatory reactions, neither P- nor E-selectin alone are sufficient to mediate eosinophil accumulation; when combined, they are essential for trafficking in ACA but are less important in the DTH reaction. Whether alpha 4 integrin-based strategies will be more effective than selectin-based strategies at inhibiting eosinophil recruitment in human disease remains to be determined.     

Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes

E-selectin is a carbohydrate-binding endothelial cell adhesion molecule that reportedly interacts with several related sialylated and fucosylated carbohydrates. The activity of leukocyte alpha1,3-fucosyltransferases (FucT-IV or FucT-VII) is an essential step in the synthesis of E-selectin ligands. Using a panel of stably transfected hemopoietic cell lines, we have investigated the role of alpha1,3-fucosyltransferases in generating E-selectin ligands, and the relationship between adhesion to E-selectin and expression of mAb-defined carbohydrates. Expression of FucT-VII was always sufficient for binding to E- and P-selectin, while the ability of FucT-IV to construct E-selectin ligands varied among different cell types. Furthermore, FucT-IV was unable to support any binding to P-selectin in a lymphoid cell line, even when expressed at levels equivalent to those in myeloid cells. FucT-IV expression generated high levels of surface Le(x)/CD15 and CDw65, whereas expression of FucT-VII correlated with a subset of mAb-defined sialyl Lewis X (sLex)-like structures. FucT-IV-associated epitopes were present on both binding and nonbinding cells, whereas all cells that expressed FucT-VII-associated epitopes bound E-selectin. However, treatment of HL60 cells with neuraminidase destroyed FucT-VII-associated epitopes at a faster rate than E-selectin binding sites. Surface expression of a subset of mAb-defined sLex-like carbohydrates is therefore a good marker for high levels of FucT-VII activity, but these carbohydrates are not themselves required for recognition of E-selectin.     

Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes

Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.     

Sialosyl-Le(a) and Sialosyl-Le(x) antigens in adhesion of cancer cells and tumor progression

Sialosyl-Le(a) and sialosyl-Le(x) are tumor-associated carbohydrate antigens present in different types of human tumors. They are commonly found on the cell surface of a variety of adenocarcinomas such as lung cancer, gastric cancer, pancreatic cancer and colorectal cancer, and in serum of cancer patients. Both antigens have been proposed as important diagnostic markers and they are used in detecting and monitoring of these diseases. Recently, it has been shown that sialosyl-Le(a) and sialosyl-Le(x) carbohydrate structures are ligands for selectins, newly described family of adhesion molecules. Selectins function as lymphocyte-homing and leukocyte enrollment receptors, or as activation dependent cell surface receptors of platelets and endothelial cells. Several lines of evidence suggest that sialosyl-Le(a) and sialosyl-Le(x) are responsible for adhesion of human cancer cells to endothelium. It has been shown that E-selectin and P-selectin present on endothelial cells mediate these interactions. The mentioned facts suggest that selectins and their carbohydrate ligands can play an important role in a selective homing of tumor cells during metastasis.     

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues

When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.     

L-selectin-mediated lymphocyte rolling on MAdCAM-1

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.     

Direct-exchange arthroplasty for the treatment of infection after total hip replacement. An average ten-year follow-up

The recirculation of naive lymphocytes from blood to lymph that is initiated in high endothelial venules (HEV) of secondary lymphoid organs such as lymph nodes and Peyer's patches (PP) is regulated by multiple interactions of adhesion receptor/counter-receptor pairs involving both selectins and integrins. We showed previously that blocking of only L-selectin is sufficient to ablate trafficking of naive CD4 cells and the development of their responses in peripheral lymph nodes but not in PP where alpha4beta7 integrins are thought to primarily regulate entry. However, although antibody to alpha4 integrins partially inhibited homing of naive CD4 cells to PP and not to lymph nodes, there was no effect on the development primary responses in these tissues or spleens. Since previous studies indicate that both alpha4beta7 integrins and L-selectin regulate adhesion of naive cells to PP HEV, we examined the effect a blockade of both adhesion pathways on the recirculation of naive CD4 cells. There was no detectable homing of naive CD4 cells to PP or lymph nodes when interactions with both receptors were inhibited, resulting in a profound depletion of naive CD4 cells and loss of antigen responses in these sites. In contrast, increased numbers of naive CD4 cells and responses of higher magnitude were found in the spleen. The results demonstrate recirculation of naive CD4 cells through tissues where entry is controlled through HEV is essential for the local generation of primary responses.     

Core 2 oligosaccharide biosynthesis distinguishes between selectin ligands essential for leukocyte homing and inflammation

Mammalian serine/threonine-linked oligosaccharides (O-glycans) are commonly synthesized with the Golgi enzyme core 2 beta-1,6-N-acetylglucosaminyltransferase (C2 GlcNAcT). Core 2 O-glycans have been hypothesized to be essential for mucin production and selectin ligand biosynthesis. We report that mice lacking C2 GlcNAcT exhibit a restricted phenotype with neutrophilia and a partial deficiency of selectin ligands. Loss of core 2 oligosaccharides reduces neutrophil rolling on substrata bearing E-, L-, and P-selectins and neutrophil recruitment to sites of inflammation. However, the diminished presence of L-selectin ligands on lymph node high endothelial venules does not affect lymphocyte homing. These studies indicate that core 2 oligosaccharide biosynthesis segregates the physiologic roles of selectins and reveal a function for the C2 GlcNAcT in myeloid homeostasis and inflammation.     

Lake Vera revisited: parity and survival rates of Anopheles punctipennis at the site of a malaria outbreak in the Sierra Nevada foothills of California

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.     

Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions

The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti-4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.     

Regulation of B cell apoptosis by Bcl-2 and Bcl-XL and its role in the development of autoimmune diseases (Review)

Leukocyte rolling is the earliest observable even in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewis(x) (sLe(x)) is required for function. Despite findings that sLe(x) inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin-dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNF alpha)-stimulated mouse cremaster is increased following treatment with either sLe(x) or the sLe(x)-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti-P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin-dependent) rolling is unaffected by either sLe(x) or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.     

A subset of CD4+ T cells expressing early activation antigen CD69 in murine lupus: possible abnormal regulatory role for cytokine imbalance

Systemic lupus erythematosus (SLE), which spontaneously develops in (NZB (New Zealand Black) x NZW (New Zealand White)) F1 mice, is strictly dependent on CD4+ T cells. We found that in these mice with overt SLE, CD4+ T cells expressing CD69 molecules, an early activation Ag, are dramatically increased in peripheral lymphoid tissues and inflammatory infiltrates in the kidney and lung, but not in peripheral blood, while CD8+ and NK1.1+ T cells were virtually CD69-. Various adhesion molecules, including LFA-1, ICAM-1, CD43, CD44, P-selectin, and E-selectin, were up-regulated. Analysis of the TCR repertoire showed no skewed TCR Vbeta usage. Studies on in vitro cytokine production of spleen cells on TCR cross-linking indicated that compared with findings in young mice, the aged mice showed severely impaired production of IL-2, IL-3, and IL-4, whereas the levels of IL-10 and IFN-gamma remained relatively intact. FACS-sorted CD69-CD4+ T cells from aged mice produced substantial amounts of these cytokines, including IL-2, IL-3, and IL-4, whereas CD69+CD4+ T cells were poor producers. Intriguingly, when cocultured, CD69+CD4+ T cells significantly inhibited the production of IL-2 by CD69-CD4+ T cells. IL-2 production by spleen cells from young mice was also markedly inhibited in the presence of CD69+CD4+ T cells obtained from aged mice. We propose that CD69+CD4+ T cells that are continuously activated by self peptides bound to MHC class II molecules in (NZB x NZW)F1 mice may be involved in the pathogenesis of SLE through abnormal regulatory effects on cytokine balance.