concanavalin-a and Hemorrhage

Reduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-gamma were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from T(H)1 to T(H)2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression.

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; CD28 Antigens; CD4-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Concanavalin A; CTLA-4 Antigen; Cytokines; Enzyme Activation; Hemorrhage; Immune Tolerance; Lectins, C-Type; Lymphocyte Activation; Mice; Ovalbumin; Proto-Oncogene Proteins c-akt; Signal Transduction; Spleen; Wounds and Injuries

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6-8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H.

Topics: Animals; Bone Marrow Cells; Cell Proliferation; Cell Survival; Concanavalin A; Cytokines; Enzyme Activation; Hemorrhage; Lidocaine; Lipopolysaccharides; Male; Mice; Mitogen-Activated Protein Kinases; Spleen; Wounds and Injuries

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL(+/+) and transgenic (PRL(-/-)) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL(-/-) mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL(+/+) mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL(+/+) mice. Splenocyte proliferation in male PRL(-/-) mice was significantly lower than in PRL(+/+) mice after T-H. Marginal differences between PRL(+/+) and PRL(-/-) mice were observed in the release of IL-2 and IFN-gamma by splenocytes, while the release of IL-10 was significantly higher in PRL(-/-) than in PRL(+/+) mice. A significant observation of our study is the release of a approximately 25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL(+/+) and PRL(-/-) mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL(-/-) transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice.

Topics: Animals; Cell Proliferation; Cells, Cultured; Concanavalin A; Cytokines; Female; Hemorrhage; Male; Mice; Mice, Transgenic; Proestrus; Prolactin; Spleen

Although diltiazem improves immune responses after hemorrhage and resuscitation, its mechanism remains unknown. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mmHg, maintained at that level for 1 h, and then adequately resuscitated and treated with diltiazem (400 micrograms/kg body wt) or saline (vehicle). One hour after hemorrhage and resuscitation, splenic microvascular blood flow was determined by laser Doppler flowmetry. Splenocytes were also harvested and ATP levels and cytoplasmic free Ca2+ concentration ([Ca2+]i) were determined by 31P nuclear magnetic resonance and fluo 3 flow cytometry, respectively. Splenocyte functions were determined by measuring interleukin (IL)-1, IL-2, IL-3, IL-6, and tumor necrosis factor concentrations in concanavalin A-stimulated supernatant with cytokine-specific cellular assays. Hemorrhage and resuscitation caused a significant decrease in the splenocyte's ability to release cytokines, which was correlated with significant reductions in splenocyte ATP levels and splenic microvascular blood flow as well as a significant increase in splenocyte [Ca2+]i. Diltiazem significantly decreased splenocyte [Ca2+]i while improving splenocyte ATP levels, cytokine production, and splenic microvascular blood flow. Nitroglycerin (71 micrograms/kg body wt) administration improved splenic microvascular blood flow to diltiazem-treated levels but failed to concomitantly improve splenocyte ATP levels or cytokine production. Thus diltiazem's immunoprotective effects appear to be the result of decreased Ca(2+)-induced splenocyte injury.

Topics: Adenosine Triphosphate; Animals; Blood Pressure; Calcium; Concanavalin A; Cytokines; Diltiazem; Hemorrhage; Immune System; Magnesium; Male; Mice; Mice, Inbred C3H; Microcirculation; Resuscitation; Sodium; Spleen

Although hemorrhage depresses splenocyte (SPL) functions and increases susceptibility to sepsis, it is not known whether increased tumor necrosis factor (TNF) or prostaglandin (PG) production are responsible for it. To study this, mice (C3H/HeN) were bled to a mean blood pressure of 35 mm Hg, maintained at that pressure for 60 min, resuscitated, and treated with ibuprofen (1.0 mg/kg body weight) or vehicle (saline). Hemorrhage reduced (P less than 0.05) SPL proliferation by 60%, SPL release of interleukin-2 (IL-2) by 47%, interferon-gamma (IFN-gamma) by 67%, TNF by 54%, and interleukin-6 (IL-6) by 46% compared to sham. In addition, splenic macrophage (sM phi) release of interleukin-1 (IL-1) and TNF was decreased by 58 and 67% (P less than 0.05), respectively. However, ibuprofen treatment increased (P less than 0.05) SPL proliferation, lymphokine (IL-2, IFN-gamma, and IL-6) synthesis, and IL-1 release by sM phi compared to hemorrhage alone. Furthermore, ibuprofen enhanced the release of TNF by SPL (+175%, P less than 0.05) and sM phi (+68%) compared to the vehicle group. Ibuprofen also decreased (P = 0.011) the susceptibility to sepsis following hemorrhage. These results indicate that PGs are involved in hemorrhage-induced suppression of cellular immunity and in the increased mortality of such animals following a septic challenge.

Topics: Animals; Blood Pressure; Cells, Cultured; Concanavalin A; Cytokines; Disease Susceptibility; DNA Replication; Hemorrhage; Ibuprofen; Immunity, Cellular; Interferon-gamma; Interleukin-2; Interleukin-6; Lymphocyte Activation; Male; Mice; Mice, Inbred C3H; Sepsis; Spleen; Tumor Necrosis Factor-alpha

Abnormalities in immune response play a major role in the increased susceptibility to infection after hemorrhage and trauma. Several studies have shown decreased release in vitro of interleukin-2 (IL-2) following blood loss. To better define in vivo the interactions between T and B cells, as well as the effects of treatment with the T cell-derived cytokines IL-2 and IL-4, mice were injected with concanavalin A at predetermined times posthemorrhage, and the percentages and numbers of splenic plasma cells producing antibody to the bacterial polysaccharide antigen levan (from Aerobacter levanicum) were determined. Decreased numbers and percentages of levan specific splenic plasma cells were found in animals treated with concanavalin A both immediately and 2 to 4 days after hemorrhage. Treatment in vivo with recombinant IL-2, but not IL-4 or anti-IL-2 receptor antibodies, following blood loss was able to increase the numbers of levan specific plasma cells to levels as high or higher than those found in normal, unhemorrhaged animals, but was unable to affect the decreased percentage of levan specific splenic plasma cells. These results suggest that the use in vivo of IL-2 may restore bacterial antigen specific antibody responses to normal levels after blood loss.

Topics: Animals; Antibodies, Monoclonal; Concanavalin A; Hemorrhage; Interleukin-2; Interleukin-4; Male; Mice; Mice, Inbred BALB C; Plasma Cells; Polysaccharides, Bacterial; Receptors, Interleukin-2; Recombinant Proteins; Spleen; T-Lymphocytes

Hemorrhage has been shown to produce abnormalities in lymphocyte function, particularly in the proliferative response to mitogens such as PHA and Con A. In order to better examine the hemorrhage-induced alterations in immune function, we determined the effects of blood loss in mice without any surgical manipulation on lymphocyte populations and subpopulations, cellular activation, and lymphokine production. Hemorrhage induced no changes in cell numbers in the spleen, thymus, lymph nodes, and bone marrow. No alterations in the relative percentages of B (B220+, mu+) and T (Lyt-1+, Lyt-2+, T3+, L3T4+) cell subpopulations were found in any organ after blood loss. Significant decreases in splenocyte proliferation in response to Con A, IL-2R expression and blast formation occurred after hemorrhage. IFN-gamma production increased 24 and 48 h post hemorrhage. Decreases in IL-2, IL-3, and IL-5 generation were present 2 h after blood loss. IL-2 production remained significantly decreased for 48 h posthemorrhage, then increased to more than twice normal levels 72 h posthemorrhage, and subsequently returned to prehemorrhage values. These results demonstrate that hemorrhage produces widespread alterations in immune function without affecting lymphocyte population and subpopulation numbers.

Topics: Animals; Concanavalin A; Hemorrhage; Immunosuppression Therapy; Interferon-gamma; Interleukins; Leukocyte Count; Lymphocyte Activation; Lymphocytes; Lymphokines; Male; Mice; Mice, Inbred C3H; Receptors, Interleukin-2; Spleen

To determine whether hemorrhage without major tissue trauma can itself produce immunosuppression, the effect of hemorrhage on the lymphocyte response to T-cell mitogen in endotoxin-resistant C3H/HEJ mice was measured. The mice were bled to achieve a mean blood pressure of 35 mm Hg, maintained at that level for one hour, and then adequately resuscitated. On days 1 through 10 thereafter, the proliferative responses of the splenocytes to concanavalin A were measured and allogeneic mixed lymphocyte reaction was performed. The proliferative responses to mitogen stimulation as well as the results of mixed lymphocyte reaction studies indicated that marked immunosuppression occurred at day 1. Immunosuppression persisted for at least five days following hemorrhage, as evidenced by mitogen stimulation assay. Another group of mice was subjected to sepsis three days after hemorrhage and resuscitation. The mortalities in the sham-hemorrhage and hemorrhage groups following sepsis were 58% and 100%, respectively. Thus, a significant depression of cellular immunity occurred following simple hemorrhage despite adequate resuscitation, and this immunosuppression enhanced the susceptibility to sepsis.

Topics: Animals; Concanavalin A; Disease Models, Animal; Disease Susceptibility; Hemorrhage; Immunity, Cellular; Immunosuppression Therapy; Infections; Lymphocyte Activation; Mice; Mice, Inbred C3H; Spleen; Time Factors

Topics: Anesthesia, Inhalation; Anesthetics; Antibody Formation; Antigen-Antibody Reactions; B-Lymphocytes; Concanavalin A; DNA; Hemorrhage; Humans; Immunosuppression Therapy; Lectins; Lymphocytes; Mitogens; Statistics as Topic; Surgical Procedures, Operative; T-Lymphocytes; Time Factors; Tritium