Expression pattern of alphavbeta3 and alphavbeta5 integrin mRNA in mouse fetal gonads

The alphavbeta3 and alphavbeta5 integrins are known as transmembrane receptors capable of binding to the RGD amino acid peptide sequence. In mouse early gonadogenesis, some proteins containing the RGD sequence are deposited into extracellular space and participate in morphogenesis. We analyzed the expression patterns of the alphavbeta3 and alphavbeta5 integrins in mouse developing gonads (10.5-13.5 days post coitum) using whole-mount in situ hybridization. The alphav integrin mRNA was homogenously expressed in developing gonadal regions. On the other hand, the beta3 integrin mRNA was found only in large and round cells (presumptive germ cells), whereas beta5 integrin was localized in gonadal somatic cells, with the exception of coelomic epithelial cells. The beta3 integrin-expressed cells were determined to be primordial germ cells because the number of these cells was drastically reduced in busulfan-treated gonads. In this study, we demonstrated that the alphavbeta3 and alphavbeta5 integrins are widely localized in the mouse developing gonads and discussed their presumptive functions on mouse gonadogenesis.     

Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration

Leukocyte-endothelial cell interactions are mediated by various cell adhesion molecules. These interactions are important for leukocyte extravasation and trafficking in all domestic animal species. An initial slowing of leukocytes on the vascular endothelium is mediated by selectins. This event is followed by (1) activation of beta2 integrins after leukocyte exposure to cytokines and pro-inflammatory mediators, (2) adherence of leukocyte beta2 integrins to vascular endothelial ligands (eg, intercellular adhesion molecule-1 [ICAM-1]), (3) extravasation of leukocytes into tissues through tight junctions of endothelial cells mediated by platelet and endothelial cell adhesion molecule-1 (PECAM-1), and (4) perivascular migration through the extracellular matrix via beta1 integrins. Inhibiting excessive leukocyte egress and subsequent free radical-mediated damage caused by leukocyte components may attenuate or eliminate tissue damage. Several methods have been used to modify leukocyte infiltration in various animal models. These methods include nonspecific inhibition of pro-inflammatory mediators and adhesion molecules by nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, inhibition of cytokines and cytokine receptors, and inhibition of specific types of cell adhesion molecules, with inhibitors such as peptides and antibodies to beta2 integrins, and inhibitors of selectins, ICAMs, and vascular cell adhesion molecule-1 (VCAM-1). By understanding the cellular and molecular events in leukocyte-endothelial cell interactions, therapeutic strategies are being developed in several animal models and diseases in domestic animal species. Such therapies may have clinical benefit in the future to overcome tissue damage induced by excessive leukocyte infiltration.     

Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms. SAMPLE POPULATION: Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results. PROCEDURES: Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry. RESULTS: Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.     

Bactrian camel (Camelus bactrianus) integrins αvβ3 and αvβ6 as FMDV receptors: Molecular cloning, sequence analysis and comparison with other species

Integrins are heterodimeric adhesion receptors that participate in a variety of cell–cell and cell–extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the αv integrin family of cellular receptors, αvβ3, αvβ6, αvβ1 and αvβ8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the αv integrins from a susceptible species as viral receptors, we have cloned Bactrian camel αv, β3 and β6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin αv, β3 and β6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel αv, β3 and β6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.     

Signaling mechanism for equine neutrophil activation by immune complexes

Neutrophils (PMN) are critical host defense cells that have a role in the pathophysiology of a variety of inflammatory diseases, particularly those diseases associated with antigen-antibody immune complexes (IC) deposited in tissues. Activation of PMN by IC is most efficient if the IC are presented immobilized on a surface. Adhesion to the immobilized IC is important for subsequent activation of PMN effector functions, such as generation of reactive oxygen metabolites. Adhesion of human PMN to immobilized IC requires the expression and activation of adhesion receptors called integrins. Of the integrins expressed on PMN, the beta 2 family has been found to be of particular importance for PMN function. The mechanism of beta 2 integrin activation during adhesion to IC has been studied in human PMN, but not in equine PMN. We show here that adhesion of equine PMN to immobilized IC requires beta 2 integrins. Like adhesion, IC-induced respiratory burst activity is dependent on beta 2 integrins. Furthermore, the signaling pathway triggering beta 2 integrin-dependent adhesion of equine PMN to IC and subsequent generation of respiratory burst activity is inhibited by the specific phosphatidylinositol 3-kinase (PI3K) antagonists wortmannin and LY294002 with IC(50) (concentration at which 50% inhibition is achieved) similar to the published values for inhibition of PI3K enzymatic activity. In contrast, PMA-induced activation of beta 2 integrin-dependent adhesion and respiratory burst activity are wortmannin and LY294002 insensitive. These data demonstrate that like in human PMN, IC-induced activation of beta 2 integrins and beta 2 integrin-dependent functions in equine PMN is dependent on PI3K activity.     

Role of sugars in surface microbe-host interactions and immune reaction modulation

Sugars in the form of monosaccharides, oligosaccharides, polysaccharides and glycoconjugates (glycoproteins, glycolipids) are vital components of infecting microbes and host cells, and are involved in cell signalling associated with modulation of inflammation in all integumental structures. Indeed, sugars are the molecules most commonly involved in cell recognition and communication. In skin, they are essential to epidermal development and homeostasis. They play important roles in microbial adherence, colonization and biofilm formation, and in virulence. Two groups of pathogen recognition receptors, C-type lectins (CTL) and their receptors (CTLR), and the Toll-like receptors enable the host to recognize pathogen-associated molecular patterns (PAMPs), which are mainly glycolipids. The CTLs can recognize a wide variety of bacteria, fungi and parasites and are important in phagocytosis and endocytosis. TLRs are expressed on the surfaces of a variety of cells, including keratinocytes, dendritic cells, monocytes and macrophages; they play a major role in innate immunity. Interaction of TLRs with PAMPs initiates a cascade of events leading to production of reactive oxygen intermediates, cytokines and chemokines, and promotes inflammation. Exogenous sugars can block carbohydrate receptors and competitively displace bacteria from attachment to cells, including keratinocytes. Thus sugars may provide valuable adjunctive anti-inflammatory and/or antimicrobial treatment. A promising approach is the use of a panel of carbohydrate derivatives with anti-adhesive efficacy against bacteria frequently involved in diseases affecting skin and other epithelia. More complete characterization of sugar receptors and their ligands will provide further keys to use of carbohydrates in immunomodulation and infection control in skin.     

The role of glycoprotein H of equine herpesviruses 1 and 4 (EHV-1 and EHV-4) in cellular host range and integrin binding

ABSTRACT: Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to alpha4beta1 or alpha4beta7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that alpha4beta1 and alpha4beta7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.     

Analysis of integrins and vascular endothelial growth factor isoforms mRNA expression in the canine uterus during perimplantation period

Integrins are the major receptors within the extracellular matrix (ECM) that mediate several functions connected with cell life and metabolism, such as cell adhesion, migration, cytoskeletal organization, proliferation, survival, and differentiation. A vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. It has been suggested that the expression of this gene may play crucial physiological roles in reproductive organs. All investigated endometrial tissues were isolated on day 10-12 after mating. Control bitches, used in this study, were in metestrus, which was determined according to the vaginal cytology and progesterone level in blood. Early pregnancy was verified by flushing the uterine horns with PBS. Total RNA was isolated from the bitches endometrium by means of the Chomczyński and Sacchi method, treated by DNase I, and reverse-transcribed into cDNA. A quantitative analysis of integrins alpha2b, beta2 and beta3, VEGF 164, 182 and 188 cDNA was performed by RT-PCR. In results we have shown an increased expression of all investigated genes (integrins alpha2b, beta2 and beta3, VEGF 164, 182, and 188) in pregnant bitches uterus as compared to non-pregnant females (P < 0.001). Our results indicated that the expression of genes encoding integrins and vascular endothelial growth factors is different in relation to the time of the embryo implantation and it is increased in the first period of this process. This may be associated with the induction of specific mechanisms responsible for receptivity of uterus following the embryo attachment. In addition, all of investigated genes are up-regulated in a pregnancy-specific manner and the increased expression of these genes may regulate the uterus function during the implantation of canine embryos.     

The role of integrins in corneal wound healing

Integrins are cell adhesion molecules important in cell–cell and cell–extracellular matrix interactions. These interactions are vital to numerous physiological processes including corneal wound healing. This review discusses the structure of integrins as well as the various roles that integrins play in the corneal wound healing process. Integrin profile abnormalities identified in various corneal pathological conditions are also reviewed.     

Adhesion receptor CD11b/CD18 contributes to neutrophil diapedesis across the bovine blood-milk barrier

epithelium. Neutrophil migration across mammary arterial endothelial cells was almost completely dependent on CD18, the beta-chain of the beta(2) integrins, and to a lesser extent on CD11b, one of the alpha-chains of the beta(2) integrins. Neutrophil migration across collagen was partially blocked by monoclonal antibodies to CD18. No inhibition was observed by monoclonal antibodies to CD11b. Conversely, neutrophil diapedesis across mammary epithelial cells was dependent to a greater extent on CD11b. These results provide evidence for different CD11b/CD18-dependent mechanisms for neutrophil diapedesis across the various cell layers of the blood-milk barrier.     

Tissue tropism of avian reoviruses is genetically determined.

Two genome segments, M2 and S1, were preferentially selected in reassortants isolated in Vero cells. Analysis with monoclonal antibodies (MAbs) against RAM-1 strain showed that the 39-kDa protein encoded by the genome segment S1 contained epitopes involved in neutralisation of virus infectivity for both Vero and chicken kidney (CK) cells. The 39-kDa protein appeared to have two major epitopes that are attachment sites for cell receptors, one interacting only with CK cell receptors and the other with both CK and Vero cell receptors but principally Vero cell receptors. These results suggest that the strain RAM-1 may have developed an epitope for Vero cell receptors owing to mutation in the S1 genome segment, but still retained the epitope responsible for infection of CK cells.     

Cell surface immunoglobulin regulated checkpoints in chicken B cell development

The bursa of Fabricius is critical for the normal development of B lymphocytes in avian species. Productive colonization of bursal follicles by B cell precursors requires surface immunoglobulin expression. We have shown using retroviral gene transfer that expression of chimeric receptors containing the extracellular and transmembrane domains of murine CD8alpha and CD8beta fused to the cytoplasmic domains of chicken Igalpha and Igbeta can support productive bursal colonization in the chicken embryo in bursal cells lacking the expression of endogenous sIgM. We show here that chimeric receptor expression does not support continued bursal cell development after hatch. However intrabursal administration of anti-CD8 antibodies that ligate the CD8alpha:Igalpha chimeric receptor results in maintained numbers of bursal cells that express the chimeric receptor in the absence of endogenous sIgM. These results support a model in which sIgM receptor expression is required for productive bursal colonization in the chick embryo but sIgM receptor ligation is required to support later B cell development after hatch.     

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.     

Role of yeast cell wall polysaccharides in pig nutrition and health protection

Polysaccharides are the major components of the yeast cell wall and play multiple functions, ranging from the carriers of immunochemical specificity and marker molecules, by which cells recognize each other and interact with the environment, to the skeletal substances that define stability, shape, and morphology of the cell. In Saccharomyces cerevisiae, the two major polysaccharides, constituting up to 90% of the cell wall dry weight, are -d-mannan and β-d-glucan, which have remarkable properties to interact with the immune system of the host. Modulation of mucosal immunity by the binding of these two polysaccharides to the specific receptors of immune cells provides beneficial effects on animal health and resistance to diseases. Specific commercial yeast cell wall polysaccharides supplied in feed (Bio-Mos®, Alltech Inc.) are able to block fimbriae of pathogenic bacteria, and thus prevent their adhesion to the mucous epithelium. Since adhesion presents the first step in microbial invasion, blocking of the receptors may prevent or eliminate infection. Yeast cell wall polysaccharides are also able to adsorb mycotoxins, thus decreasing their toxic effect and mediating their removal from the organism. Commercial yeast polysaccharides (MTB100®, Alltech Inc.) have been shown to absorb a wide range of mycotoxins at low inclusion levels. Thus, especially if the ban on antibiotic growth promoters becomes global, use of yeast polysaccharides as natural growth stimulators becomes a very urgent and rewarding issue.     

The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals

Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.