concanavalin-a and Wounds-and-Injuries

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

The depression in cell-mediated immune function following trauma-hemorrhage is shown to be restored by 17beta-estradiol (E2) administration. However, it remains unknown which of the two estrogen-receptors, (ER)-alpha or ER-beta, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER-beta(-/-) transgenic mice [knockout (KO)] and corresponding ovariectomized wild-type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and treated with E2 (50 microg/25 g) or ER-alpha agonist propyl pyrazole triol (PPT; 50 microg/25 g) following trauma-hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma-hemorrhage resulted in a significant increase in plasma tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma-hemorrhage produced a significant reduction in systemic TNF-alpha and IL-6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma-hemorrhage by macrophages and splenocyte was also prevented in E2- and PPT-treated WT mice, the release of cytokines by macrophages and splenocytes in E2- and PPT-treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma-hemorrhage.

Topics: Animals; Concanavalin A; Cytokines; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Interleukin-10; Interleukin-6; Lipopolysaccharides; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovariectomy; Phenols; Pyrazoles; Shock, Hemorrhagic; Spleen; T-Lymphocytes; Tumor Necrosis Factor-alpha; 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

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.

Topics: Animals; CD3 Complex; Concanavalin A; Drug Synergism; Estradiol; Female; Flutamide; Interferon-gamma; Interleukin-10; Interleukin-6; Lipopolysaccharides; Macrophages; Metestrus; Mice; Mice, Inbred C57BL; Shock, Hemorrhagic; Spleen; T-Lymphocytes; Tumor Necrosis Factor-alpha; Wounds and Injuries

Acute and chronic inflammation-induced expression of sialyl LewisX has already been shown to occur on alpha1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to alpha1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on alpha1-antichymotrypsin and on haptoglobin. The level of expression of sialyl LewisX on these proteins was lower than on alpha1-acid glycoprotein, in all likelihood because alpha1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl LewisX was detectable on alpha1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated LewisX was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the a3-linked fucose residues are present as sialyl LewisX on alpha1-acid glycoprotein, alpha1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for alpha1-acid glycoprotein. This leads us to propose that alpha1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.

Topics: Acute-Phase Proteins; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Antibodies, Monoclonal; Arthritis, Rheumatoid; Blotting, Western; Concanavalin A; Electrophoresis; Haptoglobins; Humans; Inflammation; Lectins; Liver; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Wounds and Injuries

To determine whether trauma induces an increase in the concentration of circulating transforming growth factor-beta (TGF-beta), and whether there is a temporal correlation between plasma TGF-beta concentration and the development of posttrauma cellular immunosuppression.. Male Sprague-Dawley rats were anesthetized, subjected to bilateral femur fractures or sham injury, and killed 1, 3, or 5 days later. Plasma TGF-beta levels, splenocyte phenotypes, mitogen-induced proliferation, interleukin-2 (IL-2) production, and IL-2 receptor (IL-2R) expression were determined at each time point.. Splenocyte proliferation increased on day 1 postinjury without corresponding change in IL-2 or plasma TGF-beta levels. Splenocyte proliferation and IL-2 production were suppressed on day 3 postinjury, while plasma TGF-beta levels peaked. No differences were observed between trauma and control groups on day 5. Splenocyte phenotypes and IL-2R expression were similar in injured and control rats at all times.. Suppression of lymphocyte proliferation and IL-2 production after trauma occurs concomitantly with a rise in plasma TGF-beta. The immune response is restored with normalization of TGF-beta concentration. These observations suggest that TGF-beta may contribute to posttrauma immunosuppression.

Topics: Animals; Cell Division; Concanavalin A; Immune Tolerance; Immunity, Cellular; Interleukin-2; Lymphocytes; Male; Pokeweed Mitogens; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-2; Spleen; Transforming Growth Factor beta; Wounds and Injuries

The purpose of this study was to try to elucidate a possible biobehavioral mechanism associated with decreased immune function in trauma patients by determining whether there is an interaction between the effects of ACTH, a stress hormone, and TGF beta, a cytokine, on peripheral blood lymphocyte proliferation. Peripheral mononuclear lymphocytes (PMLs) from healthy donors were preincubated with varying concentrations of ACTH for 24 hr, stimulated with concanavalin A and increasing concentrations of TGF beta, and incubated for 72 hr. Proliferation was assayed by tritiated thymidine incorporation. A parallel aliquot of PMLs were incubated in the presence of ACTH to determine the direct effect of ACTH on mononuclear cell TGF beta production. While harvested supernatant from cells incubated in the presence of ACTH did not contain any detectable TGF beta, ACTH as well as TGF beta were found to significantly decrease cellular proliferation independent of one another. An even greater decrease in cellular proliferation was found when both ACTH and TGF beta were used, compared to either ACTH or TGF beta alone. These results suggest a biobehavioral interaction between ACTH and TGF beta at the cellular level and that interactions to relieve stress may assist in improving function and recovery from trauma.

Topics: Adrenocorticotropic Hormone; Analysis of Variance; Burns; Cell Division; Cells, Cultured; Concanavalin A; Dose-Response Relationship, Drug; Drug Interactions; Humans; Monocytes; Psychoneuroimmunology; Reference Values; Transforming Growth Factor beta; Wounds and Injuries

Immune competence declines following major injury, and predisposes the trauma patient to infection. Interleukin-10 (IL-10), although an immunosuppressive cytokine, is also important in the initiation of immune responses. This study investigated alterations in IL-10 and immune function associated with polymicrobial sepsis following trauma using murine femur fracture (FFx) and cecal ligation/puncture (CLP) models. Mice were randomized to Normal, FFx, Alcohol and FFx (EtOH + FFx), CLP, FFx + CLP, and EtOH + FFx + CLP. Polymicrobial sepsis was induced by performing CLP 4 days after FFx, and animals were killed 14 days later; immune function was assessed by in vitro splenocyte cultures. Lymphocyte proliferative responses were significantly suppressed in FFx and CLP animals. Splenocyte IL-10 production was significantly reduced in FFx and CLP animals, with concurrent increases in nitrite and tumor necrosis factor release. This study documents that trauma induces alterations in the inflammatory cytokine cascade that affect the immune response to subsequent septic challenges.

Topics: Animals; Body Weight; Cell Division; Concanavalin A; Immunity, Cellular; Interleukin-10; Lipopolysaccharides; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Nitrites; Sepsis; Spleen; Thymidine; Tumor Necrosis Factor-alpha; Wounds and Injuries

Traumatic injury often results in profound immunopathology that can lead to immunosuppression, thereby increasing the morbidity and mortality due to sepsis. The isolation and partial characterization of an immunosuppressive glycopeptide (SAP) from serum of severely burned patients has previously been reported by our laboratory. Recently, this trauma peptide has also been identified in the serum of patients with multiple blunt trauma. This glycopeptide is capable of suppressing neutrophil chemotaxis, T-cell blastogenesis and the lysis of human erythrocytes. We demonstrate in this report that SAP inhibits interleukin 2 (IL-2) biosynthesis by mitogen-stimulated peripheral blood mononuclear cells. Peptide concentrations of 50 nmol and above significantly inhibited IL-2 production. Inhibition was not reduced by the addition of indomethacin or anti-PGE2 to cultures containing greater than 100 nmol of peptide, suggesting that inhibition is not entirely prostaglandin-mediated. Preliminary studies have shown that IL-2 suppression by SAP can be partially reversed by the addition of calcium ionophore. These results suggest a potential immunosuppressive mechanism of the trauma peptide in which T cell blastogenesis is inhibited by interference in IL-2 biosynthesis.

Topics: Calcimycin; Concanavalin A; Glycopeptides; Humans; Interleukin-2; Lymphocyte Activation; Prostaglandins E; T-Lymphocytes; Wounds and Injuries

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.

Topics: Animals; Cell Movement; Concanavalin A; Enzyme-Linked Immunosorbent Assay; Fluorescein-5-isothiocyanate; Fluoresceins; Hindlimb; Male; Salamandridae; Skin; Skin Physiological Phenomena; Wounds and Injuries

In vitro lymphocyte response to antigens, mitogens and in mixed lymphocyte culture were studied at intervals after injury in 31 patients with extensive trauma. Mean responses were significantly depressed up to 15 to 20 days. Responses were lower and the duration of suppression longer in those patients who become infected, and the suppression of response preceded the onset of infection. Extremely low responses were found in three patients who later died. This in vitro system is suitable for the serial monitoring of patients, as it reflects the extent of injury, infectious sequelae and prognosis. Its results are quantifiable and avoid the problems associated with repeated skin testing.

Topics: Accidents, Traffic; Adult; Concanavalin A; Histocompatibility Antigens Class II; Humans; In Vitro Techniques; Infections; Lymphocytes; Mitogens; Mumps; Phytohemagglutinins; Risk; Streptodornase and Streptokinase; Time Factors; Wounds and Injuries

This study was designed to investigate the relationship of in vitro and in vivo components of host immunocompetence and various biochemical parameters of injury. Thirteen multiple trauma victims were evaluated within 2 to 3 days postinjury while maintained on 5% glucose. The mean nitrogen balance of the patients was -18 gm/24 hr and the resting metabolic expenditure was increased 22.3%. Seventy-five per cent of the patients skin tested were anergic or relatively anergic. Patient lymphocyte response to the T cell mitogens phytohemagglutinin and concanavalin A were suppressed 45 and 48%, respectively, when compared to 24 normal healthy individuals. Total lymphocyte count (1,558) and percentage of T lymphocytes (63%) were within normal limits. The suppression of lymphocyte response to mitogens correlated by regression analysis with the negative nitrogen balances and resting metabolic expenditures in these patients (p less than 0.05). The depression of lymphocyte activity can be correlated with the catabolic response of injury in multiple trauma patients.

Topics: Adolescent; Adult; Concanavalin A; Female; Humans; Immune Tolerance; Lymphocytes; Male; Middle Aged; Nitrogen; Phytohemagglutinins; Random Allocation; Skin Tests; T-Lymphocytes; Wounds and Injuries

Topics: Animals; Arginine; Concanavalin A; Femoral Fractures; In Vitro Techniques; Male; Organ Size; Phytohemagglutinins; Protein Biosynthesis; Rats; T-Lymphocytes; Thymus Gland; Wounds and Injuries