Effects of weekly regrouping of prepartum dairy cows on innate immune response and antibody concentration.

Objectives were to evaluate the effects of a stable prepartum grouping strategy on innate immune parameters, antibody concentration, and cortisol and haptoglobin concentrations of Jersey cows. Cows (253±3 d of gestation) were paired by gestation length and assigned randomly to the stable (all-in-all-out; AIAO) or traditional (TRD) treatment. In the AIAO treatment, groups of 44 cows were moved into a pen where they remained for 5 wk, whereas in the TRD treatment, approximately 10 cows were moved into a pen weekly to maintain stocking density (44 cows for 48 headlocks). Pens were identical in size and design and each pen received each treatment a total of 3 times (6 replicates; AIAO, n=259; TRD, n=308). A subgroup of cows (n=34/treatment) was selected on wk 1 of each replicate from which blood was sampled weekly from d -14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, hemogram, cortisol and glucose concentrations, and haptoglobin concentration. Another subgroup of cows (n=40/treatment) selected on wk 1 of each replicate was treated with chicken egg ovalbumin on d -21, -7, and 7 and had blood sampled weekly from d -21 to 21 for determination of immunoglobulin G anti-ovalbumin. All cows (n=149) had blood sampled weekly for nonesterified fatty acid (NEFA) and ß-hydroxybutyrate (BHBA) concentrations from d -21 to 21. Treatment did not affect percentage of PMNL positive for phagocytosis and oxidative burst (AIAO=64.3±2.9 vs. TRD=64.3±2.9%) and intensity of phagocytosis [AIAO=2,910.82±405.99 vs. TRD=2,981.52±406.87 geometric mean fluorescence intensity (GMFI)] and oxidative burst (AIAO=7,667.99±678.29 vs. TRD=7,742.70±682.91 GMFI). Similarly, treatment did not affect the percentage of PMNL expressing CD18 (AIAO=96.3±0.7 vs. TRD=97.8±0.7%) and L-selectin (AIAO=44.1±2.8 vs. TRD=45.1±2.8%) or the intensity of expression of CD18 (AIAO=3,496.2±396.5 vs. TRD=3,598.5±396.9 GMFI) and L-selectin (AIAO=949.8±22.0 vs. TRD=940.4±22.3 GMFI). Concentration of immunoglobulin G anti-ovalbumin was not affected by treatment (AIAO=0.98±0.05 vs. TRD=0.98±0.05 OD). The percentage of leukocytes classified as granulocyte (AIAO=38.9±1.5 vs. TRD 38.2±1.5%) and the granulocyte:lymphocyte ratio (AIAO=0.75±0.04 vs. TRD=0.75±0.04) were not affected by treatment. Concentrations of cortisol (AIAO=14.95±1.73 vs. TRD=18.07±1.73 ng/mL), glucose (AIAO=57.6±1.5 vs. TRD=60.0±1.5 ng/mL), and haptoglobin (AIAO=3.09±0.48 vs. TRD=3.51±0.49 OD) were not affected by treatment. According to the current experiment, a stable prepartum grouping strategy does not improve innate immune parameters or antibody concentration compared with weekly prepartum regrouping.

In vivo activation of chicken macrophages by infectious bursal disease virus.

Infectious bursal disease virus (IBDV) infects and replicates in the dividing B lymphocytes of chickens. In the present study, the in vivo effect of IBDV infection on chicken macrophage populations and macrophage activation were examined. Specific-pathogen-free chickens were exposed to virulent IBDV and splenic macrophages were recovered during the acute phase of the disease. At 3 and 5 days post-infection (dpi), spleens of virus-exposed chickens had fewer macrophages than those of virus-free controls (p < 0.05). Confocal microscopic examination revealed cells that were positive for both KUL01 (macrophage surface marker) and R63 (IBDVVP2), indicating presence of the virus in macrophages. MQ-NCSU cells, an avian macrophage cell line, were susceptible to replication of IBDV. In addition, splenic macrophages were activated and had temporarily increased levels of mRNA transcripts of pro-inflammatory mediators, including IL-1beta, IL-6, IL-18, and iNOS. The robust expression of proinflammatory cytokine transcripts, along with a decrease in macrophage numbers, suggest that IBDV activates and may lead to a reduction of resident macrophages in vivo.

Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18.

To map the site involved in Mannheimia haemolytica leukotoxin (LktA) binding and biological activity within bovine CD18, bovine x human CD18 chimeric constructs were generated and coexpressed with bovine CD11a in K562 cells. Studies with the chimeric leukocyte function-associated antigen 1 transductants demonstrate that the site required for LktA binding and biological effects resides within amino acid residues 500 and 600 of the extracellular region of bovine CD18.

Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain.

This paper describes a model of tumor-induced bone destruction and hyperalgesia produced by implantation of fibrosarcoma cells into the mouse calcaneus bone. Histological examination indicates that tumor cells adhere to the bone edge as early as post-implantation day (PID) 3, but osteolysis does not begin until PID 6, correlating with the development of hyperalgesia. C3H/He mice exhibit a reproducible hyperalgesia to mechanical and cold stimuli between PID 6 and 16. These behaviors are present but significantly reduced with subcutaneous implantation that does not involve bone. Systemic administration of morphine (ED(50) 9.0 mg/kg) dose-dependently attenuated the mechanical hyperalgesia. In contrast, bone destruction and hypersensitivity were not evident in mice implanted with melanoma tumors or a paraffin mass of similar size. A novel microperfusion technique was used to identify elevated levels of the putative algogen endothelin (ET) in perfusates collected from the tumor sites of hyperalgesic mice between PID 7 and 12. Increased ET was evident in microperfusates from fibrosarcoma tumor-implanted mice but not from melanoma tumor-implanted mice, which are not hyperalgesic. Intraplantar injection of ET-1 in naive and, to a greater extent, fibrosarcoma tumor-bearing mice produced spontaneous pain behaviors, suggesting that ET-1 activates primary afferent fibers. Intraplantar but not systemic injection of the ET-A receptor antagonist BQ-123 partially blocked tumor-associated mechanical hyperalgesia, indicating that ET-1 contributes to tumor-induced nociception. This model provides a unique approach for quantifying the behavioral, biochemical, and electrophysiological consequences of tumor-nerve interactions.

Theiler's virus-infected L-selectin-deficient mice have decreased infiltration of CD8(+) T lymphocytes in central nervous system but clear the virus.

Mice with targeted deletion of L-selectin gene (L-sel(-/-)) were used to investigate the role of adhesion molecule in immunologic responses following virus infection in the central nervous system (CNS). L-Sel(-/-) mice from a resistant H-2(b) genetic background and parental wild-type H-2(b) (C57BL/6) mice were infected with Theiler's murine encephalomyelitis virus (TMEV) intracerebrally and the kinetics of virus replication and infiltration of immune cells in the CNS determined. The levels of infectious TMEV, as measured by plaque assay at 3, 7, 14, and 28 days after infection were between 4 and 6 log(10) PFU of virus per gram of CNS tissues at days 3 and 7 post-infection, and then decreased to undetectable levels by day 14 after infection in both strains of mice. The L-sel(-/-) mice had decreased numbers of CD8(+) T lymphocytes (17.72%+/-2.4) infiltrating into the CNS at 7 days post-infection when compared to wild-type mice (31.02%+/-7.5). In addition, the L-sel(-/-) mice had significantly lower levels of TMEV-specific serum IgG resulting in lower virus neutralizing activity of the serum when compared to wild-type mice. However, the L-sel(-/-) mice had 2.5-fold increase in B lymphocytes in the CNS (8.29%+/-1.1) when compared to wild-type mice (3.2%+/-0.4). Taken together, these data indicate that L-selectin plays a role in recruitment of B and CD8(+) T lymphocytes into the CNS following virus infection, which, however, did not affect the ability of the mice to clear TMEV infection.

The cytoplasmic domain of L-selectin participates in regulating L-selectin endoproteolysis.

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.

Opioid receptors on bone marrow neutrophils modulate chemotaxis and CD11b/CD18 expression.

Opiates impair neutrophil-mediated host defense, but the involvement of kappa-opioid receptors in this action has not been defined. The selective kappa-opioid receptor agonist [trans-(+)3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate inhibited macrophage inflammatory protein-2-induced chemotaxis of bone marrow neutrophils from C57BL/6 mice. Its effects were concentration-dependent (pIC(50)=10.40+/-0.61) and inhibited by naloxone (K(e)=0.27 nM). The kappa-opioid receptor agonists bremazocine and ICI-204, 488 also inhibited chemotaxis, as did the respective mu- and delta-opioid receptor agonists [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]enkephalin and [D-Pen(2,5)]enkephalin albeit with lower potencies. U-50,488H also decreased neutrophil expression of the beta(2) integrin CD11b/CD18 (Mac-1) and adhesion to plastic in a naloxone-reversible manner. The results indicate that kappa-opioid receptors expressed by neutrophils rapidly modulate chemotaxis and adhesion in vitro.

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium.

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils.

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils.

The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils.

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

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.

Shear-induced platelet activation and platelet-neutrophil aggregate formation by equine platelets.

OBJECTIVES: To determine whether platelets become activated and form platelet-platelet or platelet-neutrophil aggregates, or both, when subjected to shear. SAMPLE POPULATION: Blood obtained from 3 Thoroughbreds. PROCEDURES: Blood, with PCV adjusted to 32 (low hematocrit) or 60 (high hematocrit)%, was subjected to shear rates of 11.25, 22.5, 45, 90, 225, and 750/s for 3 minutes by use of a cone-plate viscometer. Flow cytometric techniques were used to identify activated platelets, platelet-platelet aggregates, and platelet-neutrophil aggregates. RESULTS: Shear resulted in decreased platelet count, increased mean platelet volume, platelet activation, and formation of platelet-platelet and platelet-neutrophil aggregates. These changes occurred at lower shear rates in blood with high hematocrit. Platelet-neutrophil aggregate formation was inhibited by blocking P-selectin, but not CD11/CD18 receptors. CONCLUSIONS: Shear-induced platelet activation and aggregate formation occur at physiologic shear rates. CLINICAL RELEVANCE: Shear-induced platelet activation may explain the exercise-associated platelet-neutrophil aggregates observed in Thoroughbreds undergoing treadmill exercise.

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.

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.

Neutrophil-neutrophil interactions under hydrodynamic shear stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte accumulation of P-selectin in vitro.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.

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.

Neutrophil rolling altered by inhibition of L-selectin shedding in vitro.

The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.

Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor. Identification with an L-selectin-alkaline phosphatase reporter.

Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-alpha.

Cell surface P- and E-selectin support shear-dependent rolling of bovine gamma/delta T cells.

The vascular selectins P- and E-selectin are inducible adhesion proteins expressed by endothelial cells that have been shown to support shear-dependent rolling of myeloid cells. This interaction is thought to be a prerequisite event for subsequent steps, such as tight adhesion/aggregation and transendothelial cell migration, involved in the accumulation of leukocytes into tissues. Certain lymphocyte subsets have also been shown to bind the vascular selectins, but the importance of this interaction in mediating shear-dependent rolling, as described for myeloid cells, has not been demonstrated. We expand on our earlier observation that bovine gamma/delta T cells bind E-selectin by showing that this interaction leads to a reproducible rolling event in assays done under shear forces that approximate those that occur in vivo. E-selectin, expressed by L cell transfectants or cytokine-stimulated human and bovine endothelial cells, equally supports the shear-dependent rolling interaction. The lymphocyte adhesion proteins L-selectin, CD44, and CD2 do not contribute to this event. Neuraminidase treatment of the gamma/delta T cells or addition of EDTA to the assay completely blocks the rolling interaction. We further show for the first time that P-selectin expressed by thrombin-activated platelets or a soluble P-selectin/human Ig chimera specifically binds gamma/delta T cells. The P-selectin interaction is similar to the rolling event mediated by E-selectin--it requires divalent cations and sialic acid on the lymphocyte, it lacks involvement of L-selectin and CD44, and rolling occurs under physiologic shear conditions. These results provide the documentation that the vascular selectins can support shear-dependent rolling of a lymphocyte subset and that P-selectin mediates the adhesion of gamma/delta T cells.