Pseudomonas aeruginosa exoenzyme S induces transcriptional expression of proinflammatory cytokines and chemokines.

Pseudomonas aeruginosa infection of cystic fibrosis patients causes lung damage that is substantially orchestrated by cytokines. In this study, multi-gene probe analysis was used to characterize the ability of the P. aeruginosa mitogen, exoenzyme S, to induce proinflammatory and immunoregulatory cytokines and chemokines. Exoenzyme S strongly induced transcription of proinflammatory cytokines and chemokines (tumor necrosis factor alpha, interleukin-1alpha [IL-1alpha], IL-1beta, IL-6, IL-8, MIP-1alpha, MIP-1beta, MCP-1, RANTES, and I-309), modest transcription of immunoregulatory cytokines (IL-10 and IL-12p40), and weak transcription of Th1 cytokines (IL-2 and gamma interferon). The response occurred early and subsided without evolving over time. These data suggest that cells responding to exoenzyme S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis.

Exoenzyme S from Pseudomonas aeruginosa induces apoptosis in T lymphocytes.

Exoenzyme S from Pseudomonas aeruginosa is a unique T cell mitogen; it is a powerful immunostimulus that activates a large proportion of T cells, but results in delayed and reduced lymphocyte proliferation. This study was performed to explain the discrepancy between early T cell activation and subsequent proliferation. Studies revealed that exoenzyme S induced rapid and unsustained surface expression of CD69, but could not induce interleukin-2 receptor alpha (IL-2R alpha) up-regulation on T cells. IL-2 was undetectable in supernatants and addition of rIL-2 could not reverse the unresponsiveness, indicating that anergy was not involved. Exoenzyme S induced membrane phosphatidylserine translocation, DNA hypodiploidy, and DNA fragmentation, implicating apoptosis as the mechanism for the unresponsiveness. Exoenzyme S-induced apoptosis shows features of both propriocidal and death by neglect, suggesting shared characteristics of an intermediate pathway. Thus, a Pseudomonas exoproduct induces T cell apoptosis, which may contribute to the pathogenesis of Pseudomonas infections in diseases such as cystic fibrosis.

Recombinant Pseudomonas exoenzyme S and exoenzyme S from Pseudomonas aeruginosa DG1 share the ability to stimulate T lymphocyte proliferation.

Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences. We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E. coli BL21(DE3), could induce T lymphocyte activation and proliferation. Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively. Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S. These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.

Pseudomonas aeruginosa exoenzyme S stimulates murine lymphocyte proliferation in vitro.

The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients. Previous studies have established that an exoproduct of P. aeruginosa (exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response. An animal model would facilitate studies of the pathophysiologic consequences of this activation. As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined. When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated. The optimum response occurred after 2 to 3 days in culture, at 4 x 10(5) cells per well and 5.0 micrograms of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1. There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S. Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S. Thus, murine lymphocytes, like human lymphocytes, respond to P. aeruginosa exoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis of P. aeruginosa infections in CF patients.

The capsule of Cryptococcus neoformans reduces T-lymphocyte proliferation by reducing phagocytosis, which can be restored with anticapsular antibody.

Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence of C. neoformans by suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake of C. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity to C. neoformans with anticapsular antibody.

Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigen for human T lymphocytes.

Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of the P. aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P. aeruginosa pathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated. The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.