Faculty Bibliography

Invading trophoblasts form endometrial cups in the endometrium of the pregnant mare. In the present study we characterized the maternal leucocyte response to endometrial cups from their formation to their regression. The maternal leucocyte response was correlated with the stages of trophoblast development. (1) Aggregates of CD4+ and CD8+ cells were present between the migrating and differentiating endometrial cup trophoblasts and surrounding the forming endometrial cups. (2) Numbers of CD4+ cells within the mature endometrial cups were much reduced. At the periphery of the endometrial cups CD4+ and CD8+ cells were found in patchy accumulations around endometrial glands; small clusters of CD79+ B lymphocytes were present as well. (3) Scattered CD4+ and CD8+ cells were found within dying endometrial cups; areas of cell death were infiltrated with neutrophils. Large aggregates of CD4+ cells and CD8+ cells, and small but numerous clusters of CD79+ cells and eosinophils, were found outside of the dying endometrial cups. The CD4+ or CD8+ cells were mostly CD3+ T cells; some were probably macrophages which can express both of these markers in horses. The correlation between the developmental stages of the endometrial cup trophoblast and the maternal leucocyte response suggests a complicated cytokine-mediated regulatory network

The distribution of four cytokines was analyzed in the endometrium and trophoblast of the horse between Days 30 and 55 of gestation. Endometrial tissues, invasive trophoblast (chorionic girdle), and noninvasive trophoblast (chorion and allantochorion) were examined separately. Cytokine expression was determined by amplification of specific mRNA via the reverse transcriptase polymerase chain reaction (RT-PCR). Messenger RNA for interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN gamma) was detected in endometrial tissues, unstimulated peripheral blood lymphocytes, and control kidney tissue, but not in trophoblasts. leukocytes resident in the endometrium or traversing the uterus via blood vessels might be the source of these cytokines. Endometrial tissues and invasive and noninvasive trophoblasts expressed mRNA for tumor necrosis factor alpha TNF alpha). Immuonoreactive TNF alpha protein was detected in different cell types of the endometrium and in the invasive and noninvasive trophoblast. The ubiquitous expression of TNF alpha by the endometrium and trophoblasts suggests that this cytokine might have an important role in regulating placental growth and differentiation or maternal leukocyte responses to trophoblasts. IL-2, IL-4, and IFN gamma might have important immunoregulatory roles within the endometrium

The regulatory effects of phorbol myristate acetate (PMA) on the expression of the CD4 molecule on horse T cells were investigated. On both peripheral blood lymphocytes and thymocytes, PMA resulted in a rapid and transient down-regulation of equine CD4 expression, but had no such effect on the surface expression of equine CD5, CD8 or major histocompatibility complex (MHC) class I and class II molecules. Over 75% of the surface CD4 molecules per cell were lost after a 4 h exposure to PMA at 37 degrees C. The regulation of equine CD4 expression induced by PMA was temperature dependent and reversible. The PMA-mediated loss of CD4 expression was inhibited at 4 degrees C. After 24 h of exposure to PMA, CD4 molecules were re-expressed on the cell surface, even in the continued presence of PMA. These findings demonstrate that equine CD4+ T cells undergo alterations in CD4 expression in response to PMA, and suggest that the equine homolog of the CD4 molecule is regulated by PMA in a similar manner to the human CD4 molecule

The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in addition recognized antibodies identifying new CD molecules. The workshop also succeeded in fostering co-operation between laboratories around the world which study equine immunobiology. Equine CD molecules identified by the current battery of monoclonal antibodies include EqCD2, EqCD4, EqCD5, EqCD8, EqCD11a/18, EqCD13 and EqCD44. Other antibodies are markers for MHC class I and class II molecules, for B cells, granulocytes, macrophages, T cell subsets distinct from those defined by CD4 and CD8, and other sub-populations of horse leucocytes that do not have obvious counterparts in humans, rodents, or other species. Despite the progress made in the first workshop, there are still substantial gaps in the armory of reagents available to study equine leucocyte biology, and further definition of the structure, function, and genetics of the antigens identified by the workshop clusters (WC1, WC2 etc.) and other molecules of immunological importance will be a goal of future workshops. The study of equine immunobiology and resistance to disease also urgently requires the development of tools to study equine immunoglobulins and cytokines, and these needs will provide ample scope for future studies

Equine peripheral blood lymphocytes (PBL) were enriched by positive selection using panning with a mixture of monoclonal antibodies against putative equine CD4 (Equine Leucocyte Antigen Workshop antibodies WS 1 and WS 72), or CD8 molecules (Workshop antibodies WS 12, WS 49, and WS 74). RNA was extracted from CD4 enriched cells (99% purity), from CD8 enriched cells (69% purity), from peripheral blood lymphocytes, and from neonatal equine thymus. RNA extracted from equine granulocytes and from equine kidney served as negative control. The RNA was electrophoresed in agarose and transferred to nylon membranes. Northern blots were hybridized with human and mouse cDNA probes for CD4 and CD8 alpha. The human CD4 probe detected a 2.9 kb RNA transcript in equine PBL, CD4 enriched lymphocytes, and thymocytes. The human CD8 alpha probe detected a 2.0 kb transcript in RNA from equine CD8 alpha enriched lymphocytes and thymocytes, but not from PBL or CD4 enriched lymphocytes. Mouse cDNA probes for CD4 and CD8 did not react with equine RNA. Results of hybridizations using the human probes support the assignment of the CD4 and CD8 specificities to the antibodies listed above. The results also suggest that the equine CD4 and CD8 alpha genes are more closely related to the human than to the murine counterparts

Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells

The procoagulant activity (PCA) associated with equine bronchoalveolar lavage cells was determined and compared with that expressed by peripheral blood mononuclear cells and neutrophils. Lung cell preparations from horses affected with chronic pulmonary disease were included in all experiments and there was no difference in the qualitative type of response compared with lung cells which were obtained from healthy horses. Significant amounts of PCA were expressed by cells freshly procured from bronchoalveolar lavages of healthy and diseased horses. When adherent lung cells were kept in culture for some time, cell-associated PCA slightly decreased within 4 h, reached its lowest point after approximately 24 h and rose again during the second week of culture. In contrast, freshly isolated blood mononuclear cells or neutrophils expressed little PCA. Following culture for 24 h, mononuclear cells began to express increased PCA levels. Both cultivated lung cells (comprised mainly on alveolar macrophages) and blood mononuclear cells responded to LPS by dramatically increased PCA expression, whereas neutrophils showed a small augmentation of PCA on LPS stimulation. Fresh mononuclear cells and cultivated lung cells differed in their PCA response to LPS in several respects. Blood mononuclear cells were more sensitive to LPS than lung macrophages and responded to a 100-fold lower LPS concentration than the latter. Mononuclear cell-associated PCA peaked 4 h after stimulation whereas that of cultured macrophages continued to increase up to 24 h after stimulation. Lung macrophages cultured in adherence responded to LPS stimulation with a much higher PCA increase than macrophages cultured in suspension, in teflon containers. However, the culture vessel did not influence the PCA expressed by unstimulated cells. PCA expression depended to a large extent on transcription and translation, as evidenced by a 60-85% reduction of PCA in cycloheximide- or actinomycin D-treated, LPS-stimulated lung macrophages. PCA was largely cell-associated; only a small proportion of cell-associated PCA was shed into the medium. The PCA associated with mononuclear cells and with lung macrophages was tissue factor because of its dependence on clotting factor VII and its independence from clotting factor VIII. The expression of PCA by freshly isolated cells, the lower sensitivity to LPS, and the loss of PCA in the first 24 h of cultivation are indicative of in vivo activation of lung macrophages

Equine bronchoalveolar lavage (BAL) fluid collected from 70 horses and respiratory secretions (RS) obtained from 61 of these horses were evaluated cytologically and grouped according to the histological diagnosis of the lungs from which they were obtained. The histological categories included: normal lung (8 horses); pulmonary eosinophilic infiltration (9 horses); interstitial pneumonia (5 horses); pulmonary hemorrhage (5 horses); and mild (12 horses), moderate (7 horses) and severe (24 horses) chronic small airway disease. In horses with pulmonary disease, all BAL samples and all but one RS sample differed cytologically to those obtained from normal horses; however, the type and severity of the pulmonary disease could not always be determined using either BAL or RS cytology. There was a positive association between the percentage of neutrophils in BAL and the neutrophil scores in RS specimens; there was no positive association between other cell types