calcimycin and Burns

To investigate the influence of various signal transduction modulators on the splenic T lymphocytes secretion of IL-2 and IL-10 in severely scalded mice, and to explore its mechanism.. The mice were inflicted with 18% TBSA full-thickness scald by high-pressure heat vapour, and T lymphocytes were isolated from murine splenocytes through nylon wool column at 12 and 96 post-scald hours (PSH). Then the cells were divided into following groups: i. e. control, scald, scald and modulator [1 ml of 50 micromol/L PKC inhibitor ( H-7) , 30 micromol/L tetradecanoylphorbol-13-acetate (TPA) , 10micromol/L nonreceptor tyrosine protein kinase inhibitor (herbimycin) , 25 microg/ml of mitogen activated protein kinase kinase inhibitor (PD098059) , 100 nmol/L Calcium ionophore ( A23187) were added to the cells, respectively] groups. The scald group was subdivided into S1 (with scald at 12 PSH) and S2 (with scald at 96 PSH) groups. The modulator group was subdivided into modulator, S1 and modulator( the modulators were added into cells at 12 PSH) , and S2 and modulator( the modulators were added to cells at 96 PSH) groups. The influence of modulators to T lymphocyte secretion of IL-2 and IL-10 were observed.. After the addition of H-7, the IL-2 and IL-10 levels in each group were obviously lower than that in controls( P <0. 05 or 0.01) , and that in S1 and H7 group, S2 and H7 group were obviously lower than that in scald group at corresponding time-points( P <0.01). The levels of IL-10, and especially IL-2 were elevated by TPA, but they were markedly lower than that in control group after PD098059 pretreatment. The secretion of IL-2 and IL-10 was significantly suppressed by herbimycin in S1 and herbimycin, and S2 and herbimycin groups, but those in Sl and A21387[ (2 417+/-39) pg/ml, (2 793+/-25)pg/ml] , S2 and A21387 [ (921+/-50) pg/ml, (2 633+/-35)pg/ml] groups were evidently higher than those in S1[ (1 542+/-40)pg/ml, (2 390+/-15)pg/ml] , S2 [(328+/-19)pg/ml, (1 618+/-21)pg/ml,( P <0.05 or <0.01)]groups.. PKC, calcium, MAPKK and TPK play critical roles in the dysfunction of splenic T lymphocyte secretion of IL-2 and IL-10 in severely scalded mice, among which TPK and PKC are mainly targeted to IL-2 secretion, and MAPKK is targeted to IL-10 secretion. TPA and A23187 can markedly rectify the disturbance of IL-2/IL-10 secretion ratio by increasing the IL-2 secretion after scald.

Topics: Animals; Benzoquinones; Burns; Calcimycin; Calcium; Cells, Cultured; Female; Flavonoids; Interleukin-10; Interleukin-2; Lactams, Macrocyclic; Lymphocyte Activation; Male; Mice; Mice, Inbred Strains; Mitogen-Activated Protein Kinase Kinases; Protein Kinase C; Protein-Tyrosine Kinases; Rifabutin; Signal Transduction; Spleen; T-Lymphocytes; Tetradecanoylphorbol Acetate

Neutrophils (PMNs) from patients with thermal injury are dysfunctional for the CD11b/CD18-dependent functions of diapedesis, chemotaxis, and phagocytosis. The expression of CD11b/CD18 on normal PMNs is increased after an inflammatory stimulus. We proposed that CD11b/CD18 expression on burn patient PMNs would respond abnormally to inflammatory stimuli. PMNs were obtained from nonseptic burn patients during the second week after thermal injury. PMNs from burn patients incubated with lipopolysaccharide (LPS), N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, or A23187 did not increase the expression of CD11b/CD18 to the same degree exhibited by normal PMNs. This inability to increase CD11b/CD18 was not due to differences in CD14 receptor expression, LPS binding, or factors present in the serum of burn patients. The upregulation of CD35 also was decreased on burn patient PMNs. Western blot analysis revealed decreased quantities of CD11b protein in burn patient PMNs compared with normal control PMNs. The deficiency in CD11b/CD18 expression after inflammatory stimuli may explain some of the abnormalities observed in burn PMN function.

Topics: Burns; Calcimycin; CD18 Antigens; Humans; Inflammation; Lipopolysaccharide Receptors; Lipopolysaccharides; Macrophage-1 Antigen; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Receptors, Antigen; Receptors, Complement 3b; Stimulation, Chemical; Up-Regulation

Among the fundamental immunologic abnormalities induced by serious traumatic or thermal injury are alterations in T cell activation, reduced lymphocyte interleukin-2 (IL-2) production, and associated depression of T lymphocyte proliferation. This study attempts to localize the cellular mechanisms underlying abnormal IL-2 production in thermal injury.. Following National Institutes of Health guidelines, 150 A/J mice were anesthetized, subjected to a 20% full-thickness scald burn injury or sham burn, and killed at intervals from 4 to 21 days later; splenocytes were harvested for in vitro studies. For measurement of IL-2 production, cells were cultured with either concanavalin A or a combination of the phorbol ester PMA, which directly activates protein kinase C, and the calcium ionophore A23187, which increases intracellular calcium. Cytokine mRNA expression was measured by Northern blot analysis and IL-2 production by bioassay.. Both IL-2 production and IL-2 mRNA expression were consistently suppressed in concanavalin A-stimulated cells from burned mice compared with sham burns. This suppression of IL-2 and IL-2 mRNA also occurred when T cells were activated with PMA and A23187, bypassing the earlier stages of the signal transduction mechanism. IL-1 beta and tumor necrosis factor-alpha mRNA expression were consistently increased in burned animals, indicating that decreased IL-2 mRNA expression was specific to IL-2 and not representative of a global decrease in cytokine mRNA expression.. These results suggest that the principal cellular abnormalities that result in altered T cell activation and IL-2 production after thermal injury lie downstream of the initiating signal transduction events and before IL-2 gene transcription.

Topics: Animals; Burns; Calcimycin; Interleukin-1; Interleukin-2; Lymphocyte Activation; Male; Mice; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2.

Topics: Adult; Aged; Antigens, CD; Burns; Calcimycin; Calcium; CD5 Antigens; Colforsin; Humans; In Vitro Techniques; Indomethacin; Interleukin-1; Interleukin-2; Interleukin-3; Lymphocyte Activation; Middle Aged; Monocytes; Receptors, Interleukin-2; Solubility; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Type C Phospholipases

The leukotriene generation (LTB4, 20-OH-LTB4, 20-COOH-LTB4) from PMNs of severely burned patients (n = 6) was studied by reversed-phase HPLC. Granulocytes from all patients showed a decrease in leukotriene generation which only returned to normal levels when the patients recovered from their injuries. The leukotriene generation induced by different stimuli, i.e., the Ca++-ionophore A23187 (7.3 microM) or opsonized zymosan (2 mg) in the presence of exogenous arachidonic acid (60 microM) showed similar stimulation profiles. The cellular differentiation of the respective granulocyte fractions revealed that the decreased leukotriene generation was accompanied by the occurrence of immature granulocytes in the peripheral blood. Furthermore, the studies in the presence of exogenous arachidonic acid showed that the defect in leukotriene generation from granulocytes of surviving patients was due to the availability of metabolizable substrate (i.e., free arachidonic acid). Granulocytes from one nonsurviving patient showed in addition a defect in the metabolic ability of arachidonic acid to generate the respective leukotrienes. The generation of reactive oxygen species did not correlate with the observed alterations in the formation of the leukotrienes.

Topics: Adult; Aged; Aged, 80 and over; Arachidonic Acid; Arachidonic Acids; Burns; Calcimycin; Chromatography, High Pressure Liquid; Female; Free Radicals; Granulocytes; Humans; Leukotriene B4; Luminescent Measurements; Male; Middle Aged; Oxidation-Reduction; Zymosan

Previous studies in burned patients have shown an early enhanced polymorphonuclear leucocyte (PMN) generating capacity for superoxide radical (O2.-), for the arachidonic acid (AA) lipoxygenase metabolite leukotriene B4 (LTB4) and for platelet activating factor-acether (PAF). These findings have been confirmed on a burn injury rabbit model. As we have suggested a pivotal role for an exaggerated initial (less than 36-48 h) neutrophil stimulation leading to a later (greater than 72 h) immuno-depression and anergy, we tried to modulate the early phase by drug therapy. A Ginkgo biloba extract (IPS200) injected i.v. in burned rabbits greatly reduced O2.- and LTB4 generation on A23187 challenge. IPS200 includes flavonoids and other polyphenols, inhibiting either arachidonic acid metabolism or PAF receptors, and may thus exert their modulating effect on PMN function in thermal injury.

Topics: Animals; Burns; Calcimycin; Disease Models, Animal; Diterpenes; Free Radicals; Ginkgolides; Lactones; Leukotriene B4; Male; Neutrophils; Platelet Activating Factor; Rabbits; Superoxides

The Ca ionophore A23187-induced leukotriene (LT) release (LTC4, LTB4, 20-OH-LTB4, 20-COOH-LTB4) of human PMN's from severely burned patients (n = 6) was studied by reversed-phase HPLC. The patients' granulocytes demonstrated a decrease (to zero levels) in LT generation postburn. The level of generated LT's resembled that of healthy donors when the patients recovered from their trauma (after day 40 postburn). In contrast, the granulocytes of patients who finally succumbed to their injuries showed poor responsiveness over the total time. An enhanced LTC4 production by granulocytes correlated with an increase in eosinophils within the granulocyte fraction. In addition, the reduced LTB4 production was accompanied by an enhanced LTB4 metabolism to biologically less active products (omega-oxidated metabolites). Thus, the capacity of patients' PMN's to release chemotactic substances was further decreased. The onset of this PMN dysfunction correlated with the onset of invasive microbial growth as determined by the quantitative bacterial analysis of full-thickness biopsy specimens. Our data provide evidence that the altered mediator release of patients' PMN's is closely related to a depressed host defense.

Topics: Adult; Burns; Calcimycin; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Female; Humans; In Vitro Techniques; Leukotriene B4; Male; Middle Aged; Neutrophils; SRS-A

IL2 secretion in response to the T-cell mitogen Staphylococcal protein A (SPA) is significantly decreased in patients with major burns (n = 10, greater than 20% total body surface area) up to 50 days postburn in comparison with normal control (greater than 2 to 10 U/ml and 16 to 36 U/ml, respectively). Activation of protein kinase C (PK-C) and changes in [Ca++]i are both normally implicated in the production of IL2. Bypassing the requirements for mitogen-induced increases in [Ca++]i, using the cation ionophore A23187, or activating PK-C with the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate (TPA), failed to significantly restore SPA-induced IL2 production in cell cultures from burned patients. The combination of A23187 and TPA significantly (p less than 0.005-0.001) enhanced IL2 secretion in patients' cell cultures (range, 20 to greater than 64 U/ml). However, the levels of IL2 from the burned patients' cultures remained significantly lower (p less than 0.05) than those in control cultures exposed to TPA and A23187 (range, 256 to greater than 600 U/ml). Therefore, the burn-related defect in mitogen-induced IL2 secretion is only partially bypassed with cation ionophores and phorbol esters. This suggests that the abnormality in IL2 production may not be solely related to changes in PK-C activation and in [Ca++]i but may reside in other than transmembrane signalling mechanisms required for IL2 production.

Topics: Adult; Aged; Burns; Calcimycin; Calcium; Cells, Cultured; Female; Humans; Interleukin-2; Male; Middle Aged; Protein Kinase C; Receptors, Antigen, T-Cell; Receptors, Immunologic; Receptors, Interleukin-2; Staphylococcal Protein A; Stimulation, Chemical; Tetradecanoylphorbol Acetate

Leukotriene B4 release from polymorphonuclear granulocytes of severely burned patients was reduced as compared to healthy donor cells. This decrease is due to an enhanced conversion of LTB4 into the 20-hydroxy- and 20-carboxy-metabolites and further to a decreased LTB4-synthesis. In addition, studies on the exogenous LTB4-conversion revealed an unidentified compound which was derived from LTB4. Our data suggest a modulation of the enzymatic activities involved in omega-oxidation of LTB4 (isoenzymes of cytochrome P-450).

Topics: Adult; Burns; Calcimycin; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Middle Aged; Neutrophils

Topics: Adolescent; Adult; Aged; Arachidonic Acid; Arachidonic Acids; Burns; Calcimycin; Female; Humans; Immunity, Cellular; In Vitro Techniques; Male; Middle Aged; Neutrophils; Oxygen Consumption; Superoxides; T-Lymphocytes; Time Factors

Defective phagocytic cell function may partially account for the morbidity and mortality associated with thermal injury. In experimental thermal injury in the rat, small circulating blood volumes increase the difficulty in obtaining significant data. Furthermore, purification and or elicitation procedures have the potential for altering the cell surface characteristics and/or the functional response of the cell in question. We have examined the circulating neutrophils and pulmonary alveolar macrophages of anesthetized rats following a 16-20% body surface area scald injury to the shaved back. The circulating neutrophils of thermally injured rats were examined by flow cytometry following stimulation with phorbol myristate acetate (PMA) (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric cyanine dye, dipentyloxocarboxyanine and the formation of the oxidized product of 2',7'-dichlorofluorescin diacetate-loaded cells. The alveolar macrophages were examined after stimulation with PMA (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric dye, dipropylthiodicarbocyanine and the generation of superoxide production, as assessed by the superoxide dismutase inhibitable reduction of cytochrome c. Both cells exhibited a profound inhibition of cell function 4 hours after the insult, with partial return toward control values at later time points. Furthermore, the plasma of thermally injured rats, 4 hours after the burn was inhibitory to normal rat neutrophils. Fluorescent compounds suggestive of in vivo lipid peroxidation were maximally detectable at this time point. Further research is needed to establish the role of these products in the induction of phagocytic cell dysfunction.

Topics: Animals; Burns; Calcimycin; Carbocyanines; Flow Cytometry; Fluoresceins; In Vitro Techniques; Leukocyte Count; Macrophages; Neutrophils; Oxygen; Phagocytes; Plasma; Rats; Rats, Inbred Strains; Superoxides; Tetradecanoylphorbol Acetate