Lessons from caprine and ovine retrovirus infections.

Two lentiviruses that infect sheep and goats have been shown to be closely related to the human immunodeficiency virus (HIV). These ungulate lentiviruses cause a spectrum of diseases, including arthritis in their natural hosts. The molecular and cellular biology of these viruses as well as possible pathogenic mechanisms is compared to HIV-1 in order to identify common features and significant differences between the human and animals pathogens.

An evaluation of contribution derived from investigations of equine immunodeficiencies.

Following the descriptions of immunodeficiencies in horses beginning in 1973, there has been considerable effort to develop methods for differential diagnosis and to determine the cause and prevalence of the disorders. In addition, the equine immunodeficiencies, especially combined immunodeficiency, have been studied from a comparative viewpoint with the goal of finding information applicable to similar diseases of children. Coincident with the development of knowledge about the immunodeficiencies per se, considerable information about several aspects of immunology has been obtained. It is the purpose of this review to focus on findings from experiments with equine immunodeficiencies concerning prenatal and neonatal immunology, lymphocyte function, secretory immunity, immunoreconstitution, graft-versus-host reactions and previously unrecognized diseases.

Production of arachidonic acid metabolites by caprine alveolar macrophages.

The in vitro release of arachidonic acid (AA) metabolites from caprine alveolar macrophages (CAM) stimulated with the calcium ionophore A23187 or opsonized zymosan was examined. Leukotriene B4 [5(S),12(R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid] was the major AA metabolite elicited with either agonist; smaller amounts of 12- and 5-mono-hydroxyeicosatetraenoic acid (HETE), and 12-hydroxyheptadecatrienoic acid (HHT) were also detected. Zymosan stimulation also caused the release of very small quantities of prostaglandins E2 and F2 alpha, and thromboxane B2. Our report is the first to describe arachidonic acid metabolism in CAM.

Injury induced by Trypanosoma congolense adhesion to cell membranes.

Trypanosoma congolense binds to erythrocytes and the walls of the microvasculature. Experiments were conducted to determine if the attachment of T. congolense, alone or in combination with antitrypanosome antibody, was damaging to host cells. Bovine erythrocytes were labelled with 51Cr and incubated with T. congolense to promote adhesion. Plasma from the same donor as the red blood cells was added to the erythrocyte-trypanosome aggregates and the release of 51Cr measured. There was a two- to threefold increase in 51Cr release when trypanosomes were lysed by antibody-complement interaction following adhesion to the erythrocyte. The erythrocytes were not damaged by trypanosome binding in the absence of antibody or complement. A similar mechanism may operate in vivo because experiments demonstrated an increased vascular permeability of mesenteric vessels, a site of T. congolense attachment to the microcirculation. These results suggest that the adhesion of T. congolense to host cells, followed by an immune response to the parasite, may damage the infected host by "innocent bystander" mechanisms.

Contribution of various host factors to resistance to experimentally induced bacterial endotoxemia in calves.

The reactions of 15 calves to IV administered bacterial lipopolysaccharide was investigated and was correlated with the capacity of 2 host defense mechanisms. The calves had a wide range of changes in clinical response, total WBC count, plasma lactate dehydrogenase (LD) activity, reaction to intradermally inoculated lipid A, and rectal temperature response. The early rectal temperature response of an individual calf was correlated with plasma LD activity, indicating a relationship between cell damage and fever. The concentration of antibody against lipid A at the time of lipopolysaccharide inoculation was negatively correlated with the rectal temperature changes recorded during endotoxemia, suggesting a protective ability of antibody. The capacity of a plasma inhibitor of lipopolysaccharide-mediated clotting of limulus amebocyte lysate was correlated with increases in plasma LD activity. Therefore, antibody to lipid A probably is involved in protection of cattle during endotoxemia, but the plasma lipopolysaccharide inhibitor actually may potentiate lipopolysaccharide toxicity.

Isolation and identification of equine lymphocytes and monocytes.

Various cell populations of equine mononuclear leukocytes were identified and isolated. Mononuclear leukocytes were concentrated by isopyknic centrifugation, using a solution of Ficoll and Hypaque. Three additional techniques were explored to separate monocytes from lymphocytes, and 3 methods were used to separate lymphocyte types. Cytochemical techniques for the detection of nonspecific esterase readily distinguished equine monocytes from lymphocytes. Peripheral blood lymphocytes were separated into at least 2 populations. One population had surface traits identical to thymocytes [ie, they readily bound peanut agglutinin, but lacked receptors for complement or immunoglobulin (Ig) and did not have surface Ig, as detected by immunohistochemical techniques]. This population could be isolated, using nylon-wool columns, or by depletion of complement- and Ig-binding cells during centrifugation. The other class of lymphocytes had equine complement receptors, Ig receptors, and detectable surface Ig, but was not bound by peanut agglutinin. Using rosetting techniques followed by centrifugation, this latter population was enriched. These studies provided means of isolating and detecting equine monocytes, B lymphocytes, and T lymphocytes.

Collection and cultivation in vitro of equine mammary macrophages.

Equine macrophages were obtained from female Shetland ponies by injection of Escherichia coli lipopolysaccharide through the lactiferous ducts of the mammary gland. After 6 to 11 days, balanced salt solution was injected into the mammary gland to wash out accumulated cells. Harvested cells contained a mixture of macrophages, lymphocytes, and neutrophils, with the majority of the cells of mononuclear type. In culture, cells adherent after 24 hours were characterized as macrophages by morphologic features, nonspecific esterase staining, and by the presence of complement and immunoglobulin receptors. These cultures were grown to a variety of culture media. A basal medium, consisting of 15% equine serum and 10% bovine fetal serum in conjunction with RPMI 1640 medium containing 20 mM HEPES buffer, was the most effective for maintaining spreading and adhesion of cells. Conditioned medium from mouse fibroblast cultures (L cells), added at 30% to the basal medium, improved cell monolayers by reducing giant cell formation and prolonging cell adhesion.

Stimulation and killing of bovine mononuclear leukocytes by bacterial lipopolysaccharide (endotoxin).

Bovine adherent mononuclear leukocytes were incubated with bacterial lipopolysaccharides (LPS) in vitro, and these cells produced a factor that increased the blastogenic reaction of mouse thymocytes to concanavalin A. This factor most resembles interleukin 1. The LPS were also cytotoxic for bovine adherent mononuclear leukocytes in a dose-and time-dependent manner. Cytotoxicosis was determined by the release of cytoplasmic lactic dehydrogenase. This cytotoxicosis was blocked by treating the cells with corticosteroids. Variation in the reaction to LPS occurred in cells collected from the same cow on different days and from cells collected from different cows.

Cellular and lipopolysaccharide subunit requirements for the caprine lymphoblastogenic response to endotoxin.

Caprine B lymphocytes were established as the cell type that divided when cultured with lipopolysaccharide (LPS), and lipid A was defined as the in vitro mitogenic component of LPS. The conclusion that the caprine B lymphocyte was stimulated by LPS was based on 3 observations: (i) Numbers of B lymphocytes increased in cultures containing LPS, but not in unstimulated or concanavalin A-stimulated cultures, (ii) mixtures of T lymphocytes and monocytes did not incorporate tritiated thymidine when LPS was added, and (iii) removal of monocytes from mixtures of T and B lymphocytes did not reduce the LPS-stimulated reaction. Stimulation of B lymphocytes by LPS occurred when less than 1% monocytes were present and was augmented by greater than 5% monocytes. The lipid A subunit of LPS was most likely responsible for mitogenesis, since purified lipid A stimulated lymphocytes and the addition of polymyxin B, a specific inhibitor of lipid A activity, blocked the lymphocyte reaction.

Lymphocyte subpopulations of the goat: isolation and identification.

Experiments were designed to identify and isolate goat monocytes and lymphocytes. Leukocytes were obtained from peripheral blood, thymus, spleen, and lymph nodes. Thymocytes lacked surface immunoglobulin (Ig) detectable by immunohistochemical techniques and receptors for complement or Ig. A majority of the thymocytes nd nylon-wool purified blood T lymphocytes bound peanut agglutinin (PNA). However, a distinct minority of the T lymphocytes lacked receptors for PNA. The PNA nonreactive lymphocytes represented a greater portion of the cells of the spleen and lymph nodes, suggesting that caprine T lymphocytes may be placed in PNA reactive and nonreactive subpopulations. Lymphocytes with complement and Ig receptors and surface Ig were detected in the blood, spleen, and lymph nodes. These cells met the criteria for caprine B lymphocytes. The lymphocytes were enriched by immunocytoadherence techniques followed by isopyknic centrifugation, and the portion of PNA reactive cells decreased as the number of B lymphocytes increased.

Regulation by antibody of lectin-induced lymphocyte proliferation: antibody inhibition of mitogenesis and release of lectin from cell surfaces.

Studies were conducted to determine the ways in which antibody regulates proliferation of lymphocytes stimulated by phytohemagglutinin (PHA) and concanavalin A (ConA). Equine lymphocytes were reacted with PHA or ConA for 1 to 10 hours, washed free of excess lectin, then cultured for 3 days and examined for proliferation and incorporation of [3H]thymidine. Both PHA and ConA induced lymphocytes to proliferate after short incubation periods. This response could be interrupted by the addition of antibody to the respective lectin. Total suppression of the proliferative response occurred if antibody was added within the first 2 hours after initial contact with PHA or ConA. Addition at times up to 48 hours produced either total or partial suppression. The removal of excess antibody 4 hours after addition to the culture did not reverse the suppression. Using radiolabeled ConA and PHA, it was observed that suppressive antibody inhibited the release of lectin from metabolically active lymphocytes and from surfaces where pinocytosis was not possible or was minimized. Antibody did not affect the rate of degradation of lectin by the cells. These results indicate that antibody attached to lectins, already bound to the lymphocyte surface, can terminate the lymphocyte response and retard the rate of release of lectin from the cell membrane.

Experimental infection of sheep by caprine arthritis-encephalitis virus and goats by progressive pneumonia virus.

The lentiviruses, caprine arthritis-encephalitis virus (CAEV) and progressive pneumonia virus (PPV) of sheep, cause major diseases in their respective hosts; however, the infectivity of these viruses for closely related species has not been determined. Experiments were conducted to determine whether CAEV would infect sheep and whether PPV would infect goats. Upon inoculation with CAEV, lambs developed a nonsuppurative arthritis and antibody to CAEV, and the virus was isolated up to 4 months later. Exposure of 3 lambs to CAEV-infected adult goats did not lead to demonstrable infection after 18 months. Young goats inoculated with PPV replicated the virus and developed arthritis and antiviral antibody. These results demonstrate that these distinctly different lentiviruses may infect and cause diseases in species other than their accustomed host. Presently used techniques may not be effective in differentiating which lentivirus is responsible for infection of sheep and goats. Our results also indicate that mixing sheep and goats may adversely influence attempts to eradicate lentiviruses from these species.

In vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes from ponies.

The in vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes (PBL) from ponies were studied. Prednisolone inhibited lymphocyte stimulation by phytohemagglutin (PHA) in a dose-dependent manner, without inducing lysis even at large doses. The PBL from horses heterozygous for the combined immunodeficiency trait responded to corticosteroid treatment the same as did PBL from normal ponies. Removal of the corticosteroid after incubation with PBL from normal ponies partially restored responsiveness of these cells to PHA. Chronic in vivo treatment of ponies with corticosteroids caused a marked decrease in the absolute numbers of circulating lymphocytes. Most remaining lymphocytes had detectable surface immunoglobulin and C3 receptors, suggesting a greater decrease in the T-lymphocyte population. In spite of this, there was little change in the in vitro PHA- or keyhole limpet hemocyanin-sensitized ponies. In general, the corticosteroid effects of lysis, as well as the mitogenic and antigenic responses of PBL from ponies, were similar to those previously reported for human lymphocytes.

Agammaglobulinemia in a horse with evidence of functional T lymphocytes.

Agammaglobulinemia was diagnosed in a 1-year-old Thoroughbred horse on the basis of the following observations: (1) absence of serum immunoglobulins M, A, and G(T); (2) small amounts of serum immunoglobulin G (16 mg/100 ml); (3) absence of specific antibody in the serum of the horse following immunization and challenge exposure to 2 antigens; (4) absence of plasma cells, primary follicles, and germinal centers in a lymph node removed after antigenic stimulation; (5) absence of "natural" serum antibodies to rabbit-erythrocytes which were easily detectable in age-matched control horse serums; and (6) increased susceptibility to infections. There was evidence of functional cell-mediated immunity which included a skin response to injected phytolectins, skin response to antigen challenge following sensitization, and in vitro proliferative response of lymph node cells to phytohemagglutinin. An intact cell-mediated immune response was also supported by the observation that the horse lived to 17 months of age without antibody production, whereas horses with an absence of both antibody production and cell-mediated immunity (combined immunodeficiency) die by 4 months of age without immunologic intervention. The known features of agammaglobulinemia in this horse are similar to those in sex-linked agammaglobulinemia in persons and are unique among the immunodeficiences described in other animals.

Augmented T lymphocyte responses and abnormal B lymphocyte numbers in goats chronically infected with the retrovirus causing caprine arthritis-encephalitis.

Caprine arthritis-encephalitis is a retrovirus-induced disease resulting in lymphoproliferative lesions of the CNS and joints. Peripheral blood leukocytes of chronically infected goats were analyzed for the types of cells present and for their reactivity to viral antigen and polyclonal stimulants. Two of 9 infected goats had abnormal numbers of B lymphocytes--one elevated and the other deficient. Lymphocyte reactivity to viral antigens was transiently detectable by a lymphoblastogenic assay in 5 of the 9 goats. The reactive cells were peanut agglutinin-negative T lymphocytes. Concanavalin A induced more division in T lymphocytes of infected goats than in lymphocytes of noninfected goats, whereas the reactions to phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide were no different in the 2 goat groups. It is concluded that goats infected by the caprine arthritis-encephalitis virus have antigen-reactive T lymphocytes and that infection promotes the response to a nonspecific T-cell stimulant.

Hypogammaglobulinemia predisposing to infection in foals.

Measurement of serum immunoglobulins in 46 foals less than 2 weeks old revealed 9 foals with hypogammaglobulinemia. The hypogammaglobulinemia was attributed to failure in transfer of immunoglobulins from dam to foal via colostrum. Three of the affected foals did not nurse at all, or only slightly, and 2 of these died of infections within a few days after birth, whereas the 3rd foal did not grow as well as normal foals. Six of the affected foals nursed in an apparently normal manner, and 5 of these had nonfatal respiratory infections between 2 and 5 weeks of age. Analysis of serum samples from surviving foals demonstrated that immunoglobulins were eventually produced. One other foal examined had hypogammaglobulinemia at 57 days of age, an age when the foal should have produced large amounts of immunoglobulin independent of passive transfer. This foal had simultaneous infections and hypogammaglobulinemia, but eventually produced normal amounts of immunoglobulin. Cellmediated immunity was normal at 3 months of age. This condition was designated transient hypogammaglobulinemia and was thought to be due to a temporary inability to make immunoglobulins.