Identity of activation molecule 3 on superantigen-stimulated bovine cells is CD26.

Most CD8(+) T cells in cultures of bovine mononuclear cells stimulated with staphylococcal enterotoxin C1 develop an unusual phenotype characterized by expression of activation molecule 3 (ACT3). This superantigen-dependent phenotype may be relevant to immunopathogenesis mediated by certain microbial toxins. The size and N-terminal sequence of immunoprecipitated ACT3 indicate that ACT3 is the bovine orthologue of CD26.

Bovine mammary immune response to an experimental intramammary infection with a Staphylococcus aureus strain containing a gene for staphylococcal enterotoxin C1.

Staphylococcal enterotoxin C (SEC), a superantigen, is the most frequently expressed enterotoxin by bovine strains of Staphylococcus aureus causing mastitis. To examine the possible impact of SEC on the immune response of the bovine mammary gland, we monitored changes in lymphocyte subpopulations in mammary glands of four lactating cows after intramammary instillation of S. aureus strain Rn4220 transformed with a plasmid containing a gene coding for SEC1. Four other lactating cows received the same strain transformed with the plasmid without the SEC1 gene (positive control), and four cows were untreated (negative control). Mammary quarter milk samples for somatic cell count (SCC) analysis and determination of N-acetyl-beta-D-glucosimindase (NAGase) activity levels were collected daily for 21 d postinstillation. Flow cytometry utilizing three-color analysis was used to phenotype lymphocyte subpopulations isolated from milk samples collected on d 0, 4, 7, 11, 14, 18, and 21 postinstillation from all the cows. Milk from mammary gland halves (positive control and experimental) or all mammary quarters (negative control) was collected for flow cytometric analysis. Increased NAGase activity, SCC, and isolated S. aureus demonstrated that infection was established in mammary quarters intrammarily instilled with bacteria. There were no significant differences (P > 0.05) in the proportions of BoCD4 helper T lymphocytes or BoCD8 cytotoxic T lymphocytes between the two infected treatment groups. There was a significant day x treatment difference of the proportion of a gammadelta T cell subpopulation that did not express BoCD2, but did express the ACT2 activation molecule and a significant treatment difference of a gammadelta T cell subpopulation that expressed BoCD2, but not the ACT2 activation molecule (P < 0.05). Results do not support the hypothesis that the presence of the gene for SEC1 alters the mammary BoCD4 or BoCD8 T lymphocyte response to infection.

Antiviral activity of shiga toxin 1: suppression of bovine leukemia virus-related spontaneous lymphocyte proliferation.

Human infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemorrhagic colitis. The Stxs belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Many RIPs have potent antiviral activity for the plants that synthesize them. STEC strains, both virulent and nonvirulent to humans, are frequently isolated from healthy cattle. Interestingly, despite intensive investigations, it is not known why cattle carry STEC. We tested the hypothesis that Stx has antiviral properties for bovine viruses by assessing the impact of Stx type 1 (Stx1) on bovine peripheral blood mononuclear cells (PBMC) from cows infected with bovine leukemia virus (BLV). PBMC from BLV-positive animals invariably displayed spontaneous lymphocyte proliferation (SLP) in vitro. Stx1 or the toxin A subunit (Stx1A) strongly inhibited SLP. Toxin only weakly reduced the pokeweed mitogen- or interleukin-2-induced proliferation of PBMC from normal (BLV-negative) cows and had no effect on concanavalin A-induced proliferation. The toxin activity in PBMC from BLV-positive cattle was selective for viral SLP and did not abrogate cell response to pokeweed mitogen- or interleukin-2-induced proliferation. Antibody to virus or Stx1A was most effective at inhibiting SLP if administered at the start of cell culture, indicating that both reagents likely interfere with BLV-dependent initiation of SLP. Stx1A inhibited expression of BLV p24 protein by PBMC. A well-defined mutant Stx1A (E167D) that has decreased catalytic activity was not effective at inhibiting SLP, suggesting the inhibition of protein synthesis is likely the mechanism of toxin antiviral activity. Our data suggest that Stx has potent antiviral activity and may serve an important role in BLV-infected cattle by inhibiting BLV replication and thus slowing the progression of infection to its malignant end stage.

Unique superantigen activity of staphylococcal exfoliative toxins.

Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vbeta2 or several murine Vbetas. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vbetas (huVbetas), although, neither toxin expanded huVbeta2. ETB induced expansion of murine T cells bearing Vbetas 7 and 8, those that have the highest homology to the huVbetas expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVbetas that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported.

Induction of type 2 cytokines by a staphylococcal enterotoxin superantigen.

Persistent intramammary infections of dairy cows with Staphylococcus aureus may involve immunosuppression mediated by bacterial toxins such as enterotoxins and other super-antigens (SAgs). Previously we found that stimulation of bovine PBMC with staphylococcal enterotoxin C (SEC) induced a unique phenotype of activated CD8+ T cells expressing a newly identified activation molecule, ACT3. In the present study we found that SEC induced the expression of interleukin (IL)-4 and IL-10 mRNAs, two cytokines associated with type 2 responses. Elevated levels of IL-4 and IL-10, observed between day 0 and day 4 of culture, were associated with temporary inhibition of proliferative responses of T cells, evidenced by a decrease in numbers of CD4+ T cells and a small increase in numbers of CD8+ T cells. Vigorous proliferation of T cells occurred between days 4 and 7 of culture and with a bias towards CD8+ T cells. Acquisition of the ACT3+ phenotype by CD8+ T cells was preceded by induction of IL-4 mRNA. Thus, in the bovine system, SAgs may hinder protective responses by inducing type 2 cytokines, which interfere with immune clearance of many microbial pathogens. The results of the study are consistent with the hypothesis that SAgs are involved in immunosuppression, and suggest possible immunomodulatory mechanisms.

Activation of bovine lymphocyte subpopulations by staphylococcal enterotoxin C.

Staphylococcus aureus is a major mastitis-causing pathogen in cattle. The chronic nature of bovine staphylococcal mastitis suggests that some products or components of S. aureus may interfere with the development of protective immunity. One class of molecules that could be involved are superantigens (SAgs). Although a significant number of mastitis isolates produce SAgs, the effect of these molecules on the bovine immune system is unresolved. To determine if immunosuppression caused by SAgs could play a role in pathogenesis, we monitored bovine lymphocytes exposed to staphylococcal enterotoxin C1 (SEC1). Activation of bovine lymphocytes by either SEC1 or concanavalin A (ConA) was influenced by the gammadelta/alphabeta T-cell ratio in the culture. Compared to ConA-induced stimulation, cultures stimulated with SEC1 generated small numbers of CD4+ alphabeta T cells expressing high levels of interleukin-2 receptor alpha chain (IL-2R alpha) and major histocompatibility complex class II (MHCII), suggesting that SAg exposure does not lead to full activation of these cells. This state of partial activation was most pronounced in cultures with a high gammadelta/alphabeta ratio. In contrast, significant numbers of CD8+ alphabeta T cells expressed high levels of IL-2R alpha and MHCII, regardless of the gammadelta/alphabeta ratio and the stimulant used. CD8+ blasts in cultures stimulated with SEC1 also expressed another activation marker, ACT3, previously detected predominantly on thymocytes and CD4+ T cells. Although gammadelta CD2- and CD2+ T cells expressed MHCII and IL-2R alpha following stimulation with SEC1, only a few cells increased to blast size, suggesting that they were only partially activated. The results suggest ways in which SAgs might facilitate immunosuppression that promotes the persistence of bacteria in cattle and contributes to chronic intramammary infection.

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

Bronchoalveolar leucocyte responses to trickle infections with Nippostrongylus brasiliensis in rats.

There is a need for experimental systems allowing study of host responses generated by continuous, low-level exposures to parasites. To assess the pulmonary inflammatory responses in different types of exposure to infection we used bronchoalveolar lavage (BAL). Rats sensitized by 500 Nippostrongylus brasiliensis larvae given in 20 doses over a 4-wk period (group T) and challenged with 500 larvae 33 days after the initial exposure were compared to rats initially given 1 sensitizing dose of 500 larvae (group B) and also to naive, sham-treated (group S) rats, subsequently challenged with 500 larvae. BAL performed prior to final challenge revealed markedly increased numbers of macrophages and eosinophils in group T, but there were only minor changes in numbers of these cells in group B. After final challenge, numbers of BAL macrophages and eosinophils were greater in group T than in group B, although in group B there was a rapid increase in numbers of these cells. Changes in numbers of BAL neutrophils were not correlated with previous sensitization to N. brasiliensis. Thus, there was a pronounced influx of leucocytes into the pulmonary lumen after secondary challenge in rats sensitized by repeated exposures to low doses of larvae.

Variations of broncho-alveolar lavage techniques affect cell recoveries.

Broncho-alveolar lavage (BAL) has become a routine method of sampling airway and alveolar milieu. Variations of BAL techniques, such as application of chest massage, may have an effect on cell yields. We compared numbers and composition of cells obtained by BAL with and without chest massage from naive rats and from rats infected 7 days earlier with 3,000 larvae of Nippostrongylus brasiliensis. Both techniques revealed significant increases in numbers of agranulocytes and granulocytes after N. brasiliensis infection, but the total yield of cells was 4-5 times greater when massage was used. The use of massage consistently increased the yield of macrophages, but did not result in consistently greater yields of lymphocytes or granulocytes.

Trickle infections with Nippostrongylus brasiliensis in rats: larval migration through the lungs.

The effects of exposure of rats to repeated low-level (trickle) infections with Nippostrongylus brasiliensis were assessed by measuring intestinal and lung worm burdens. Worm recoveries from the intestine, made during a period of trickle infection in rats of different ages, showed a virtually complete rejection of intestinal worms in old rats and a partial rejection in young rats. Recoveries from lungs were made in young rats after challenge infection with 500 third-stage (L3) larvae, given after a 2- or 4-wk period of sensitization, during which rats were infected with 10 or 20 doses of 25 larvae. Such trickle infections elicited a strong host response to a challenge infection, manifested by low recoveries of larvae and an increased duration of larval retention in lungs. In another group of rats sensitized by a single dose of 250 L3 larvae, the recovery of larvae from challenge infection and their clearance from the lungs were similar to these observed in rats uninfected prior to challenge. The effect of trickle infections on preintestinal stages was most pronounced and consistent in rats exposed to larvae the greater numbers of times and over the longest period.