Corinna Krüger: Functional characterization of the interaction between bacterial superantigens and bovine neutrophilic granulocytes.
Bacterial superantigens are proteins which interact with cells of the immune system in a rather typical manner. They crosslink MHC class II molecules on antigen-presenting cells with T cell receptors on T cells irrespective of their antigenic specificity and resulting in a polyclonal T cell activation. The pathogenic importance of superantigens is the following dysregulation of the immune response. While classical interactions of bacterial superantigens with human and murine B or T cells have been studied intensively, the potential interactions of superantigens with other cell types have gained much less attention. Thus, in the first part, this thesis addressed the question whether superantigens of S. aureus have any direct or indirect influence on the viability of bovine neutrophilic granulocytes in vitro. The studies were based on qualitative and quantitative flow cytometric methods.
The superantigens tested (SEA, SEB) had no apparent direct effect on the viability of pure granulocytes (PMN, neutrophils and eosinophils). However, in the presence of blood mononuclear cells (MNC), superantigens led to an accelerated death of neutrophils but not of eosinophils or mononuclear cells. Compared to medium controls, in superantigen-stimulated cultures only about 10-50% of the neutrophils survived after 24 hours in vitro. Accelerated death of neutrophils required the presence of at least 10% MNC. Minimal effective SEA concentrations ranged between 10-100 fg/ml (SEB 0.1-10 pg/ml). The effect could be mimicked by culture supernatants of superantigen-stimulated MNCs, suggesting that direct cell-cell interactions are not required for the killing. In order to determine the responsible factors or mediators, different strategies were chosen. Blocking monoclonal antibodies specific for tumor necrosis factor-α (TNF-α) or the Fas molecule were unable to inhibit the killing of neutrophils. Even Brefeldin A, an inhibitor of golgi transport and cytokine secretion, did not abolish the accelerated killing of neutrophils. Blocking of nitric oxide generation or PGE2 synthesis of monocytoid cells also could not alter the superantigen-induced killing of bovine neutrophils.
For these reasons it was asked whether inhibitors of caspases (enzymes of the intracellular apoptosis cascade) are able to abolish or modify this MNC-dependent killing of neutrophils. A caspase-1-inhibitor (Z-VAD-FMK, inhibits caspase-1, -3, -4
and -7) and two selective inhibitors, for the capase-2 (Ac-VDVAD-CHO) and the caspase -8 (Z-IETD-FMK), displayed no effect on the constitutive apoptosis rate of pure neutrophils. They were also unable to block the superantigen-induced killing when used alone either in cocultures of MNC and neutrophils or when introduced in neutrophil cultures treated with superantigen-conditioned MNC supernatants. However, an equimolar combination of caspase-1 and caspase-2-inhibitor resulted in a reduced superantigen-induced killing of neutrophils in MNC/neutrophil cocultures. This effect was less pronounced with neutrophils incubated with superantigen-conditioned MNC supernatants. All tested caspase inhibitors did not increase the numbers of viable MNC after 6 days in vitro, but each inhibitor blocked the SEA-induced MNC proliferation by about 50%. CD8+ cells were slightly more affected than CD4+ T cells. Taken together, superantigen-induced, released factors of activated mononuclear cells seem to kill bovine neutrophilic granulocytes and the release is sensitive to the combined action of caspase-1-like caspases and caspase-2.
S. aureus is a major pathogen for cattle, causing varying forms of subclinical and clinical mastitis. Two groups of S. aureus virulence factors (leukotoxins and superantigens) are supposed to play an important role in the pathogenesis of this disease. In order to detect all known and putative members of leukotoxins and superantigens, the above described flow cytometric approach was utilized to check whether S. aureus isolates of cows with mastitis produce leukotoxins, superantigens or both virulence factor groups.
Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro.
Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Numbers of viable PMN, the activation of MNC (based on their increase in size) and the blastogenesis-inducing potential of the supernatants were recorded flow cytometrically.
Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n = 32), operatively termed “leukotoxin supernatants”, killed already purified granulocytes (neutrophils and eosinophils) in vitro. The second group (n = 20), operatively termed “superantigen supernatants”, induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n = 16) contained neither leukotoxin nor superantigen activity. The absence of superantigen activities in leukotoxin supernatants was confirmed by heat treatment which destroyed
the leukocytotoxic activity but did not reveal any MNC-activating potential. This suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can be easily detected by the functional assay described.
The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical mastitis (leukotoxin = 41%; superantigen = 30.8%). The higher frequency of leukotoxin-producing isolates in cases of clinical mastitis (leukotoxin = 55.2%; superantigen = 27.6%) was not significant. These findings argue against the dominant role of superantigens or leukotoxins in the cause or the course of S. aureus-induced bovine mastitis. However, it remains to be clarified whether these virulence factors are expressed differentially in vitro as compared to the in vivo situation.
S. aureus utilizes leukotoxins to reduce the numbers of viable neutrophils. An influence on these cells during the early infection phase seems to make sense from the pathogens point of view. The described indirect and negative effect of superantigens for bovine neutrophils may provide new insights in pathogenic mechanisms by which superantigens modulate the hosts immune response.