Contribution of transcript stability to a conserved procyanidin-induced cytokine response in γδ T cells.

γδ T cells function in innate and adaptive immunity and are primed for secondary responses by procyanidin components of unripe apple peel (APP). In this study, we investigate the effects of APP and purified procyanidins on γδ T-cell gene expression. A microarray analysis was performed on bovine γδ T cells treated with APP; increases in transcripts encoding granulocyte-monocyte colony stimulating factor (GM-CSF), IL-8 and IL-17, but not markers of TCR stimulation such as IFNγ, were observed. Key responses were confirmed in human, mouse and bovine cells by reverse transcription-PCR and/or ELISA, indicating a conserved response to procyanidins. In vivo relevance of the cytokine response was shown in mice following intraperitoneal injection of APP, which induced production of CXCL1/KC and resulted in neutrophil influx to the blood and peritoneum. In the human T-cell line, MOLT-14, GM-CSF and IL-8 transcripts were increased and stabilized in cells treated with crude APP or purified procyanidins. The ERK1/2 MAPK pathway was activated in APP-treated cells, and necessary for transcript stabilization. Our data describe a unique γδ T-cell inflammatory response during procyanidin treatment and suggest that transcript stability mechanisms could account, at least in part, for the priming phenotype.

A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation.

The expression of cell-surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW approximately 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N-linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up-regulated staining more than 18-fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on approximately 37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that approximately 18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up-regulated on leucocyte subsets during the inflammatory response.

Antibody binding to a conformation-dependent epitope induces L-selectin association with the detergent-resistant cytoskeleton.

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37 degrees C, at 4 degrees C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.

Effects of continuous exposure to stromal cell-derived factor-1 alpha on T cell rolling and tight adhesion to monolayers of activated endothelial cells.

Immobilized stromal cell-derived factor-1 alpha (SDF-1 alpha) has been shown to induce tight adhesion of T cells to purified ICAM-1 in assays done under flow conditions. In this study, we show that soluble SDF-1 alpha induced a rapid (within 20 s) cessation of rolling and tight adhesion of >90% of the rolling T cells on monolayers of activated endothelial cells under similar flow. Within 4 min, the T cells had either started to migrate between the endothelial cells or re-entered the rolling and circulating lymphocyte pool. This deadherence of the firmly bound cells, with either ensuing transmigration or continued rolling, was most likely due to desensitization of lymphocytes to the continuously present SDF-1 alpha. The released rolling lymphocytes could still respond to other activating signals by a second round of tight adhesion. Pretreating the lymphocytes with pertussis toxin almost completely blocked the effect of the chemokine, confirming that the induction of firm adhesion was due to the function of the chemokine on the lymphocytes and not the endothelial cells. Pretreating the endothelium with SDF-1 alpha did not lead to firm adhesion of subsequently added lymphocytes, also indicating that the effect was due to soluble, not endothelially bound, chemokine. Blocking experiments showed that the same molecules mediated rolling before and after SDF-1 alpha-induced tight adhesion. This is the first study to demonstrate the effect of soluble SDF-1 alpha on T cell rolling on an endothelial cell monolayer. The data broaden our understanding of the stimulatory factors directing the firm adhesion and ensuing transmigration of leukocytes into tissues through activated endothelium.

Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods.

A prerequisite for studies on bovine neutrophils is a reliable method of neutrophil isolation from blood to obtain highly purified cell populations that are functionally active. Since current techniques of neutrophil isolation fall short of these requirements, we have developed a newer and more effective technique for isolation of bovine neutrophils that utilizes biomagnetic beads coated with a monoclonal antibody that recognizes an abundant surface antigen on bovine neutrophils to purify these cells. Comparison of the purity and viability of bovine neutrophils isolated by a conventional method (continuous Percoll density gradient) with this new method showed that neutrophils isolated with biomagnetic beads were higher in purity and had an increased yield. In addition, cells isolated with biomagnetic beads demonstrated normal or even improved function in assays of chemotaxis, phagocytosis, degranulation, and respiratory burst activity. Finally, bovine neutrophils isolated using this method showed an overall lower level of spontaneous apoptosis, which correlates well with the high level of viability observed in the purified cell preparations. Thus, this method represents a significant advance over current methods for isolating bovine neutrophils and would be widely applicable to labs studying the biochemistry and signal transduction pathways in these cells.

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.

A circulating bovine gamma delta T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin.

Tissue-specific localization of TCR-defined subsets of gamma delta T cells has been widely reported; however, the mechanisms responsible for this phenomenon are poorly understood. We describe a bovine gamma delta T cell TCR-associated subset that preferentially localizes in the spleen. This subset was characterized by coexpression of CD8, and was found to lack surface expression of E-selectin ligands, GR Ag ligands, as well as low expression of L-selectin. The CD8-positive gamma delta T cell subset did not accumulate at sites of inflammation as efficiently as CD8-negative gamma delta T cells that, in contrast, express E-selectin and GR ligands and high levels of L-selectin. This is the first demonstration of a gamma delta T cell subset, which exhibits a defined tissue tropism, having a unique adhesion molecule expression profile. These results demonstrate that in some cases tissue-specific accumulation of gamma delta T cell subsets can be predicted by expression, or lack of expression, of defined homing molecules.

Analysis of surface antigen expression and host defense function in leukocytes from calves heterozygous or homozygous for bovine leukocyte adhesion deficiency.

OBJECTIVE: To analyze surface antigen expression and functional responses of leukocytes from calves heterozygous and homozygous for bovine leukocyte adhesion deficiency (BLAD). ANIMALS: 8 clinically normal calves, 4 calves heterozygous for BLAD, and 4 calves homozygous for BLAD. PROCEDURE: Surface antigen expression was examined by flow cytometric analysis of leukocytes stained with monoclonal antibodies. Neutrophil function analyses included phagocytosis and killing of Candida albicans and measurement of respiratory burst activity using cytochrome c and dihydrorhodamine 123 assays. Differential leukocyte counts also were performed. RESULTS: Leukocytes from heterozygous calves were similar to those of clinically normal calves with respect to surface antigen expression, C albicans phagocytosis and killing, and respiratory burst activity. In contrast, neutrophils from calves homozygous for BLAD had significantly reduced phagocytic and yeast-killing capacity but had higher respiratory burst activity than cells from clinically normal or heterozygous calves. Homozygous calves also had extreme neutrophilia and significantly more immature neutrophils. CONCLUSIONS: The heterozygous BLAD genotype does not cause detectable functional differences in leukocytes, compared with those of clinically normal calves. In contrast, leukocytes from homozygous calves seem to upregulate alternative host defense capabilities (eg, respiratory burst activity) to partially compensate for the lack of typical adherence-dependent host defense functions.

Preferential tissue localization of bovine gamma delta T cell subsets defined by anti-T cell receptor for antigen antibodies.

Clonal and oligoclonal populations of gammadelta T cells, with respect to the expression of T cell receptors for antigen (Tcr), have been shown to localize in normal and inflamed tissues. The mechanisms responsible for the tissue-selective accumulation of these subsets are still not known. gammadelta T cells are the predominant T cell subset in newborn calves, making this animal a useful model to study these cells. However, molecular markers defining tissue-specific bovine ydelta T cell subsets are only now being developed. In this report, we describe three new anti-bovine gammadelta Tcr mAbs: GD3.8, GD197 and GD3.1, which provide useful tools to study these cells. GD3.8 recognized virtually all gammadelta T cells in the blood; whereas GD3.1 and GD197 recognized mutually exclusive as well as overlapping subsets. Using these three mAbs, four separate subsets of gammadelta T cells were defined: subset 1 (GD3.8+, GD3.1+, GD197-); subset 2 (GD3.8+, GD3.1-, GD197+); subset 3 (GD3.8+, GD3.1+, GD197+); and subset 4 (GD3.8+, GD3.1-, GD197-). Subset 4 constituted a minor population in the blood; however, it predominated in the spleen and, in some cases, represented a 300% increase over blood levels. The percentage of GD3.1-positive gammadelta T cells was found to be increased in experimentally inflamed lymph nodes, suggesting that subset 1 cells may be preferentially retained in or recruited to sites of inflammation. Some subset 4 cells also exhibited a decreased ability to respond to PHA. These studies demonstrate that bovine gammadelta T cell, Tcr-defined subsets, exhibit unique accumulation and activation characteristics that may provide clues to their function and regulation.

Comparative ultrastructure and expression of L-selectin on bovine alphabeta and gammadelta T cells.

Compared to most alphabeta T cells, bovine gammadelta T cells express two to five times the level of L-selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine gammadelta and alphabeta T cells and to study the expression of L-selectin on these cells. It is interesting that gammadelta T cells had more than twice as many microvilli and other surface projections on a per cell basis as alphabeta T cells. This was not due to the gammadelta T cell being larger; after adhesion and fixation procedures used for the EM studies, gammadelta T cells averaged 3.9 +/- 0.01 microm in diameter, whereas alphabeta T cells were slightly larger (5.7 +/- 0.01 microm in diameter). As previously shown for human neutrophils and lymphocytes, L-selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage-specific antigen on gammadelta T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of gammadelta T cells in various in vitro flow assays may be due to the greater number of microvilli on gammadelta T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L-selectin expression on gammadelta versus alphabeta T cells because L-selectin is clustered at the tips of microvilli.

Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism.

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.

Characterization of an adhesion molecule that mediates leukocyte rolling on 24 h cytokine- or lipopolysaccharide-stimulated bovine endothelial cells under flow conditions.

Bovine gamma/delta T cells and neutrophils roll on 24 h cytokine- or lipopolysaccharide-stimulated bovine fetal umbilical cord endothelial cells in assays done under physiological flow. An antibody directed against E- and L-selectin has minimal blocking effect on this rolling interaction. mAbs were raised against the stimulated bovine endothelial cells and screened for inhibition of gamma/delta T cell rolling. One mAb (GR113) was identified that recognizes an antigen (GR antigen) selectively expressed by stimulated bovine endothelial cells isolated from fetal umbilical cord, mesenteric lymph nodes, and aorta. GR113 blocked bovine gamma/delta T cell as well as neutrophil rolling on the 24 h-activated endothelial cells. The GR antigen was constitutively expressed at low levels on the cell surface of platelets and its expression was not upregulated after stimulation of these cells with thrombin or phorbol myristate acetate. However, stimulated platelets released a soluble, functionally active form of the molecule that selectively bound in solution to gamma/delta T cells in a mixed lymphocyte preparation. GR113 mAb blocked the binding of the soluble platelet molecule to the gamma/delta T cells. Soluble GR antigen also bound a subset of human lymphocytes. Cutaneous lymphocyte-associated antigen (CLA) bright human lymphocytes exhibited the greatest capacity to bind the GR antigen, though CLA was not required for binding. Subsets of both human CD4 and CD8 T cells bound the GR antigen. Immunoprecipitation experiments showed the GR antigen to be 110-120 kD Mr. The binding of soluble GR antigen was inhibited by EDTA and O-sialoglycoprotease, but not neuraminidase treatment of the target cells.

Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis.

Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

Comparison of E-selectin-binding glycoprotein ligands on human lymphocytes, neutrophils, and bovine gamma delta T cells.

We compared E-selectin-binding cell surface ligands on bovine gamma delta T cells and human leukocytes using an E-selectin/Ig chimera. The chimera worked well in flow cytometric studies and showed that bovine gamma delta T cells were the only lymphocyte population in newborn animals that bound E-selectin chimera. Furthermore, the chimera blocked gamma delta T cell rolling on E-selectin. Chimera reacted with four potential glycoprotein ligands of 180, 200, 250, and 300 kDa in Western blot analysis of gamma delta T cell detergent lysates, and it specifically precipitated at least two of these E-selectin ligands (200 and 250 kDa) from lysates of cell surface biotinylated gamma delta T cells. Preclearing bovine gamma delta lysates of GD3.5 Ag and workshop cluster 1 did not abrogate E-selectin ligand precipitation, suggesting that these surface markers do not represent E-selectin ligands. Human neutrophils possessed three E-selectin-binding ligands of approximately 80 to 90, 130, and 230 kDa, while human lymphocytes variably possessed three ligands of 120, approximately 220 to 240, and 260 kDa. Cross-precipitation experiments confirmed the results of others that neutrophil L-selectin serves as the 80 to 90-kDa E-selectin ligand. The human lymphocyte approximately 220 to 240-kDa and 260-kDa ligands may be analogous to the bovine gamma delta T cell molecules, whereas the 120-kDa is unique to human cells. The identities of the human and bovine lymphocyte E-selectin ligands are unknown. Finally, E-selectin ligand-1 apparently may have a minimal role, if any, in lymphocyte/E-selectin interactions, since a polyclonal anti-E-selectin ligand-1 serum stained a minimal number of cutaneous lymphocyte-associated Ag-positive human lymphocytes and bovine gamma delta T cells.

Localization of alpha/beta and gamma/delta T lymphocytes in Cryptosporidium parvum-infected tissues in naive and immune calves.

The nature of the host's T-lymphocyte population within the intestinal villi following Cryptosporidium parvum infection was characterized with a bovine model of cryptosporidiosis. In naive animals, infection with C. parvum resulted in substantial increases in the numbers of alpha/beta T cells, both CD4+ (150%) and CD8+ (60%), and of gamma/delta T cells (70%) present within the intestinal villi of the infected ileum. In immune animals, the host T-lymphocyte response to a challenge infection with C. parvum was restricted to alpha/beta T cells. The number of CD4+ T cells within the Peyer's patch of the ileum increased dramatically; however, there was little change in the number or localization of CD4+ T cells within the intestinal villi. In contrast, the number of CD8+ T cells within the intestinal villi increased following a challenge infection. In addition, the CD8+ T cells were found to be intimately associated with the epithelial cells of the intestinal villi. The precise correlation between the accumulation of CD8+ T cells and the normal site of parasite development suggests an important role for CD8+ T cells in the immune animal.

Tumor necrosis factor-alpha induces leukocyte recruitment by different mechanisms in vivo and in vitro.

It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.

gamma/delta T cell/endothelial cell interactions.

Ruminant gamma/delta T cells exhibit unique patterns of tissue- and inflammation-specific recruitment. Studies of other cells, such as alpha/beta T cells and even neutrophils, have clearly shown that interactions with the vascular endothelium regulate the entry of these cells into tissues. The leukocyte/endothelial cell interaction is a dynamic event that occurs under considerable shear force associated with blood flow, and it involves a variety of adhesion molecules expressed by both the leukocyte and endothelium. We have begun a systematic analysis of gamma/delta T cell interactions with endothelial and other cells in novel in vitro assays that reflect blood flow to gain insight into the molecular basis of the selective recruitment of these lymphocytes to epithelial-associated tissues and sites of inflammation. We have found that bovine gamma/delta T cells in newborns predominantly use the selectin family of leukocyte and vascular adhesion proteins to interact with endothelial cells. This is in direct contrast to other lymphocytes, such as alpha/beta T cells, which predominantly interact with selectins only after conversion to a memory cell phenotype. Our analyses of gamma/delta T cell-selectin interactions will be summarized.

Characterization of peptide diffusion into electropermeabilized neutrophils.

The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.

Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family.

In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1-negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross-immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function.

Analysis of bovine gamma delta T cell interactions with E-, P-, and L-selectin. Characterization of lymphocyte on lymphocyte rolling and the effects of O-glycoprotease.

In earlier studies, we found that bovine gamma delta T cells avidly bind and roll under physiologic flow on E- and P-selection. In this study, we have extended our analyses of shear interactions of gamma delta T cells and have found that once gamma delta T cells permanently bind to endothelial cell monolayers they can support continued rolling of newly arriving gamma delta T cells. Anti-L-selection Abs blocked gamma delta T cell rolling on immobilized gamma delta T cells, and it was L-selection on the rolling cell, not the bound cell, that mediated the interaction. We propose, as we have for neutrophils, that lymphocyte/lymphocyte rolling represents a mechanism for amplification of the extravasation process. Having defined P-, E-, and L-selectin interactions with gamma delta T cells, we tested the susceptibility of the ligands for these selectins to treatment with O-glycoprotease, a compound that selectively cleaves mucin-like selectin ligands. O-glycoprotease treatment of gamma delta T cells blocked their interaction with P-selectin, but not E-selectin. Rolling of O-glycoprotease-treated cells on bound untreated gamma delta T cells was unaffected; however, O-glycoprotease treatment of the bound gamma delta T cells blocked their capacity to support rolling interactions. These results define a new lymphocyte/selectin interaction that may be important in amplifying lymphocyte extravasation and suggest that gamma delta T cell ligands for P- and E-selectin are distinct, whereas ligands for P- and L-selectin may be similar.