concanavalin-a and Bacteroides-Infections

Sepsis-induced suppression in T-cell proliferation follows deranged Ca(2+) signaling in adult rats. In preliminary studies, we observed suppression in T-cell proliferation in septic neonatal rats as well. In this study, we assessed splenic T-cell cytosolic Ca(2+) concentration, [Ca(2+)](i), as its elevation plays an important role in T-cell proliferation. Also, we investigated the role of PGE(2) in sepsis-related changes in T-cell [Ca(2+)](i) in animals pretreated with cyclooxygenase-1 (COX-1) inhibitor (resveratrol) and cyclooxygenase-2 (COX-2) inhibitor (NS-398). Sepsis was induced in 15-day-old rat pups by intraperitoneal implantation of fecal pellets containing Escherichia coli and Bacteroides fragilis. The sham group consisted of pups implanted with sterile fecal pellets. Septic and sham pups were sacrificed 24 h after implantation and their spleens were removed. The spleens from sham and septic pups, along with spleens from unoperated control pups, were processed for single cell suspensions, and T cells were isolated using nylon wool columns. Fura-2 fluorophotometry was employed for the measurement of [Ca(2+)](i) (in nM units) in T cells stimulated with concanavalin A (ConA). Our results show that ConA-mediated T-cell [Ca(2+)](i) response is significantly suppressed in septic neonatal rats. Pretreatment of pups with COX-2, but not COX-1 inhibitor, prevented the decrease in the [Ca(2+)](i) response. These findings suggest that PGE(2) might induce the attenuation in T-cell Ca(2+) signaling during sepsis in neonatal rats.

Topics: Animals; Animals, Newborn; Bacteroides Infections; Calcium; Concanavalin A; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Escherichia coli Infections; Isoenzymes; Lymphocyte Activation; Membrane Proteins; Nitrobenzenes; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Resveratrol; Sepsis; Signal Transduction; Spleen; Stilbenes; Sulfonamides; T-Lymphocytes

Sepsis is associated with depressed T-cell functions and increased circulating levels of immunosuppressive agents. TGF-beta is a potential anti-inflammatory cytokine that can modify T-cell growth and differentiation. The up-regulation of TGF-beta and the mechanism of its action on the T-cells during septic injury have not been resolved. We hypothesized that in sepsis TGF-beta produced by macrophages acts on T-cells in a paracrine manner to suppress interleukin (IL)-2 production and proliferation. In this study, we examined the circulating TGF-beta levels in a rat model of Gram-negative bacterial sepsis, and compared the abilities of adherent and non-adherent splenocytes to produce TGF-beta. Additionally, we investigated the causal relationships of hrTGF-beta to concanavalin A (ConA)-induced T-cell responses and the intracellular mechanism of the generation of these responses in normal splenic rat T-cells. Sepsis was induced in rats by intraabdominally implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10000 CFU). Adherent and non-adherent splenocytes were isolated by differential adherence using Ficoll gradient centrifugation. T-cells were purified by use of Nylon wool columns. We observed a 3-6-fold increase in the circulating levels of TGF-beta in sepsis. Western blots and ELISA determinations revealed a 2.5-3-fold increase in cell-associated TGF-beta protein levels in adherent splenic cells. Northern analyses also showed a marked increase in TGF-beta mRNA expression in adherent cells during sepsis. On the other hand, a significant change was not observed in the TGF-beta protein and mRNA expression in non-adherent splenocytes. Pretreatment of control rat T-cells with hrTGF-beta decreased both ConA-induced proliferation (by 35-40%) and IL-2 mRNA expression (by > 50%). Further, whereas incubation of control rat T-cells with either ConA or TGF-beta for 24 h resulted in a 10-15-fold increase in cAMP generation, the addition of hrTGF-beta along with ConA resulted in a 50-60-fold increase in cAMP. These results suggest that in sepsis, TGF-beta produced by splenic macrophages can act in a paracrine manner on T-cells to depress their IL-2 mRNA expression, IL-2 production and proliferation after up-regulation of cAMP which can interfere with T-cell signaling for proliferation.

Topics: Animals; Bacteremia; Bacteroides fragilis; Bacteroides Infections; Cell Adhesion; Concanavalin A; Cyclic AMP; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Interleukin-2; Lymphocyte Activation; Male; Rats; Rats, Sprague-Dawley; Reference Values; Spleen; T-Lymphocytes; Transcription, Genetic; Transforming Growth Factor beta

Alterations in T-lymphocyte kinetics and/or activation have been implicated in burn and traumatic injury. This study evaluated concanavalin A (Con A) regulation of both intracellular Ca2+ (Cai2+) and proliferation in T-lymphocytes harvested from spleens of septic rats. Rats were implanted with fecal pellets containing Escherichia coli (150 colony forming units (CFU)) and Bacteroides fragilis (10(4) CFU). T-cell[Ca2+]i was measured before and after treatment of cells with ConA, using Fura-2 and microfluorophotometry. Splenic lymphocytes were cultured for 72 h with Con A to assess their proliferative response. As compared to sterile-implanted rats, the septic rat T-lymphocytes Cai2+ response to Con A significantly decreased on days 1 and 2 after implantation. A significant decrease in T-cell proliferative response to Con A, compared to sterile controls, was found in septic rats on day 2 but not on day 1. These results suggest that Con A-mediated proliferation in T-cells occurs secondarily to a decrease in cellular Ca2+ signalling. The depression in the T-cell proliferative response during sepsis could contribute to a decrease in host's resistance against sepsis.

Topics: Animals; Bacteroides fragilis; Bacteroides Infections; Calcium; Concanavalin A; Disease Models, Animal; Escherichia coli Infections; In Vitro Techniques; Kinetics; Lymphocyte Activation; Male; Rats; Rats, Sprague-Dawley; Sepsis; Signal Transduction; Spleen; T-Lymphocytes