Energetics of defective macrophage antigen presentation after hemorrhage as determined by ultraresolution 31P nuclear magnetic resonance spectrometry: restoration with adenosine triphosphate-MgCl2.

BACKGROUND. The purpose of this study was to determine whether a decrease in macrophage energetics contributes to the profound immune dysfunction that occurs after hemorrhage and, if so, whether adenosine triphosphate (ATP)-MgCl2 treatment has any beneficial effects on the above parameters. METHODS. C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg, maintained at that pressure for 60 minutes, resuscitated with their own shed blood and Ringer's lactate, and treated with ATP-MgCl2 (80 mumol/kg body weight) or saline solution (vehicle). Peritoneal macrophages were harvested 1 hour after resuscitation and ATP levels were determined by 31P nuclear magnetic resonance spectrometry. In addition, macrophage functions were determined by measuring antigen presentation capacity (AP), as well as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) synthesis. RESULTS. Hemorrhage caused a significant decrease in peritoneal macrophage AP function, as well as IL-1, IL-6, and TNF synthesis, by 52% +/- 14%, 91% +/- 12%, 78% +/- 8%, and 89% +/- 8%, respectively, which was correlated with a 78% +/- 6% decrease in macrophage ATP levels (p less than 0.05). Treatment with ATP-MgCl2 after hemorrhage restored macrophage ATP levels (p less than 0.05) and significantly increased (p less than 0.05) macrophage AP, IL-1, IL-6, and TNF release by 110% +/- 21%, 130% +/- 38%, 124% +/- 17%, and 66% +/- 24%, respectively. CONCLUSIONS. The decreased macrophage ATP levels may be the cause of defective macrophage AP and cytokine release after hemorrhage, and both macrophage ATP levels and macrophage immune functions can be restored with adjuvant ATP-MgCl2 treatment after hemorrhage.     

Blockade of prostaglandin production increases cachectin synthesis and prevents depression of macrophage functions after hemorrhagic shock.

Although hemorrhage severely depresses macrophage functions, it is not known whether the increased TNF-alpha or PGE2 production is responsible for it. To study this C3H/HeN mice were bled to mean blood pressure of 35 mmHg for 60 minutes, resuscitated, and treated with either ibuprofen (1.0 mg/kg body weight) or vehicle (saline). Hemorrhage increased plasma prostaglandin E2 (PGE2) levels by 151.7% +/- 40.0% (p less than 0.05) and significantly decreased peritoneal macrophage (pM phi) antigen presentation (AP) by 60.5% +/- 7.3%, Ia expression by 52.3% +/- 7.6%, and interleukin-1 (IL-1) synthesis by 60.5% +/- 12.3% compared to shams. However ibuprofen treatment reduced PGE2 plasma levels by 61.3% +/- 12.1% and significantly increased AP (+237.0% +/- 95.3%), Ia expression (+72.8% +/- 27.5%), IL-1 synthesis (+235.7% +/- 134.7%), and cachectin synthesis (+485.8% +/- 209.0%) compared to vehicle-treated animals. These results indicate that prostaglandins but not cachectin are involved in the suppression of pM phi functions following hemorrhage because blockade of prostaglandin synthesis improved depressed macrophage functions despite enhanced cachectin synthesis.     

Insights into the mechanisms of defective antigen presentation after hemorrhage

Although hemorrhage depresses macrophage antigen presentation (AP), a critical component in eliciting an antigen-specific immune response, it is not known which particular step in macrophage AP (i.e., uptake, ingestion, catabolism, or presentation of degraded antigens to T cells) is defective. To study this, C3H/HeN mice were bled to an arterial mean blood pressure of 35 mm Hg, maintained for 60 minutes, and then adequately resuscitated. Peritoneal and splenic macrophage cultures were prepared 2 and 24 hours after hemorrhage. Macrophage AP capacity was measured by coculturing macrophages with the T-helper cell clone D10.G4.1. To gain information about macrophage ability to digest the specific antigen conalbumin, lysosomal activity was bypassed by use of chemically denatured conalbumin peptides. To study macrophage ability to present conalbumin peptides, macrophages were fixed and D10.G4.1 proliferation in response to fragmented conalbumin was determined. AP of native conalbumin by peritoneal macrophages and splenic macrophages was depressed (p less than 0.05) by 50% (peritoneal macrophages) and 55% (splenic macrophages) 2 hours and 57% (peritoneal macrophages) and 35% (splenic macrophages) 24 hours after hemorrhage. In contrast, presentation of conalbumin peptides was only slightly decreased. In addition, the ability of fixed peritoneal macrophages and splenic macrophages to present conalbumin peptides was similar in hemorrhaged and sham mice. Because bypassing of macrophage lysosomal activity with degraded native antigens prevented the suppression of AP, the results suggest that hemorrhage-induced suppression of AP is not caused by a reduced macrophage capacity to present antigenic peptides but by decreased antigen catabolism by macrophages.     

Interferon-gamma attenuates hemorrhage-induced suppression of macrophage and splenocyte functions and decreases susceptibility to sepsis.

Although it is known that interferon-gamma synthesis and macrophage functions are depressed after hemorrhage, it remains to be determined whether systemic administration of interferon-gamma has any effect on hemorrhage-induced depression of macrophage and splenocyte functions. To study this, C3H/HEN mice were bled to a mean blood pressure of 35 mm Hg, maintained for 60 minutes, and followed by adequate fluid resuscitation. The mice then received either 1000 units interferon-gamma or saline solution (vehicle). Peritoneal (pM phi) and splenic (sM phi) macrophages and splenocytes were isolated 24 hours later. PM phi antigen presentation was measured by coculturing pM phi with the D10.G4.1 cell clone. Major histocompatibility complex class II (Ia) antigen expression was determined by direct immunofluorescence. Cytokine release by pM phi, sM phi, and splenocytes was assessed with specific bioassays. For survival studies, mice were subjected to sepsis 3 days after hemorrhage. Treatment with interferon-gamma restored (p less than or equal to 0.05) hemorrhage-induced suppression of pM phi antigen presentation capacity and Ia antigen expression and increased (p less than or equal to 0.05) interleukin-1 and tumor necrosis factor release by pM phi and sM phi, as well as splenocyte proliferation (p less than or equal to 0.05). Interferon-gamma also decreased (p less than or equal to 0.007) the susceptibility to sepsis after hemorrhage. Thus interferon-gamma represents a potent agent for treating hemorrhagic shock-induced immunosuppression and for increasing the ability of the host defense system to combat bacterial infections after hemorrhage.     

Depressed antigen presentation function and membrane interleukin-1 activity of peritoneal macrophages after laparotomy

Although major tissue trauma produces profound depression of cell-mediated immunity, it is not known whether surgical trauma (i.e., midline laparotomy) has any adverse effect on the antigen presentation function and membrane interleukin-1 (IL-1) activity of peritoneal macrophages. To study this, C3H/HEJ (endotoxin-tolerant) mice were anesthetized. An approximately 1-inch midline abdominal incision was made, followed by abdominal closure. On days 1, 3, 5, and 7, peritoneal macrophages were harvested by means of peritoneal lavage, and antigen presentation capability was tested by incubating various numbers of peritoneal macrophages with 2 X 10(4) D10.G4.1 cells per well in the presence of conalbumin (400 micrograms/ml). The T helper cell clone (D.10.G4.1) proliferates on recognition of conalbumin in the context of Iak and also proliferates in the presence of membrane-bound IL-1 plus concanavalin A. To measure membrane IL-1 expression in peritoneal macrophages, Concanavalin A (10 micrograms/ml) was substituted for conalbumin. Cultures were incubated for 72 hours, pulsed with tritiated thymidine, and harvested. Peritoneal macrophages from laparotomized mice induced significantly less T helper cell proliferation on days 1 and 3 in the antigen presentation assay (37% and 30%, respectively; p less than 0.05) and in the membrane IL-1 assay (14% and 10%, respectively; p less than 0.05) as compared with the control. This difference was not detectable on day 5. More effective antigen presentation capability (167% of control; p less than 0.05) was seen on day 7. Thus laparotomy by itself produces marked depression of both antigen presentation function and membrane IL-1 activity of peritoneal macrophages, which may enhance susceptibility to intra-abdominal sepsis.     

Sepsis-induced changes in macrophage co-stimulatory molecule expression: CD86 as a regulator of anti-inflammatory IL-10 response

BACKGROUND: Sepsis remains a substantial risk after surgery or other trauma. Macrophage dysfunction, as a component of immune suppression seen during trauma and sepsis, appears to be one of the contributing factors to morbidity and mortality. However, whereas it is known that the ability of macrophages to present antigen and express major histocompatibility complex MHC class II molecules is decreased during sepsis, it is not known to what extent this is associated with the loss of co-stimulatory receptor expression. Our objectives in this study were, therefore, to determine if the expression of co-stimulatory molecules, such as CD40, CD80, or CD86, on peritoneal/splenic/liver macrophages were altered by sepsis (cecal ligation [CL] and puncture [CLP] or necrotic tissue injury (CL) alone; and to establish the contribution of such changes to the response to septic challenge using mice that are deficient in these receptors. METHODS: To address our first objective, male C3H/HeN mice were subjected to CLP, CL, or sham (n = four to six mice/group), and the adherent macrophages were isolated from the peritoneum, spleen, or liver at 24 h post-insult. The macrophages were then analyzed by flow cytometry for their ex vivo expression of CD40, CD80, CD86, and/or MHC II. RESULTS: The expression of CD86 and MHC II, but not CD40 or CD80, were significantly decreased on peritoneal macrophages after the onset of sepsis or CL alone. In addition, CD40 expression was significantly increased in Kupffer cells after sepsis. Alternatively, splenic macrophages from septic or CL mice did not show changes in the expression of CD80, CD86, or CD40. To the degree that the loss of CD86 expression might contribute to the changes reported in macrophage function in septic mice, we subsequently examined the effects of CLP on CD86 -/- mice. Interestingly, we found that, unlike the background controls, neither the serum IL-10 concentrations nor the IL-10 release capacity of peritoneal macrophages from septic CD86 -/- mice were increased. CONCLUSION: Together, these data suggest a potential role for the co-stimulatory receptor CD86/B7-2 beyond that of simply promoting competent antigen presentation to T-cells, but also as a regulator of the anti-inflammatory IL-10 response. Such a role may implicate the latter response in the development of sepsis-induced immune dysfunction.     

Diltiazem restores IL-2, IL-3, IL-6, and IFN-gamma synthesis and decreases host susceptibility to sepsis following hemorrhage

Various beneficial effects of calcium channel blockers on cell and organ function following endotoxic shock, organ ischemia, and reperfusion have been reported; however, it is not known whether these agents have any salutary or deleterious effects on immune responses after low-flow conditions. Therefore, the aim of this study was to determine (a) the effect of hemorrhage on lymphocyte IL-2, IL-3, IL-6, and IFN-gamma synthesis, and (b) whether diltiazem has any salutary or adverse effects on these parameters when administered following hemorrhage and resuscitation. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mm Hg, maintained at that level for 60 min, and resuscitated with shed blood plus twice that volume of Ringer's lactate. Immediately following resuscitation mice received either diltiazem (2400, 800, or 400 micrograms/kg body wt), or an equivalent volume of saline. The mice were sacrificed 24 hr later, splenic lymphocytes were obtained, and their capacity to produce lymphokines was assessed. The results indicated that in the vehicle-treated animals, hemorrhage significantly decreased (P less than 0.05) IL-2, IL-3, IL-6, and IFN-gamma synthesis by 82 +/- 19%, 64 +/- 28%, 71 +/- 11%, and 86 +/- 14%, respectively. However, diltiazem (400 but not 2400 micrograms/kg) treatment after hemorrhage restored lymphocyte capacity to produce IL-2, IL-3, IL-6, and IFN-gamma (P less than 0.05). Additional groups of animals were subjected to sepsis by cecal ligation and puncture 3 days following hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage.

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.     

Effect of blood transfusion on antigen presentation function and on interleukin 2 generation

To study the effect of blood transfusion (BT) on cell-mediated immunity, we examined the antigen presentation function of peritoneal macrophages and interleukin 2 (IL-2) generation by splenocytes. C3H/HEJ mice were transfused with 0.2 mL of fresh allogeneic blood obtained from C57BL/6 mice; they were killed on days 1, 3, and 7 after BT. A second group of C3H/HEJ mice was transfused with 0.2 mL/d of the same allogeneic blood on three successive days; they were killed on day 7 following the last BT. The antigen presentation function of peritoneal macrophages was measured by utilizing a D10.G4.1 T-helper cell clone; IL-2 activity in supernatants of concanavalin A-stimulated splenocytes was tested by utilizing an IL-2-dependent HT-2 cell line. The results indicate that although antigen presentation function remains unaffected after single and multiple BTs, the ability of splenocytes to generate IL-2 decreases significantly even after a single BT. Thus, the increased susceptibility to infection and the additional immune perturbations in malignant neoplasms following BT may be due in part to decreased IL-2 generation.     

Effect of 6-gingerol on pro-inflammatory cytokine production and costimulatory molecule expression in murine peritoneal macrophages

BACKGROUND: Pro-inflammatory cytokines produced primarily by macrophages are key elements in many surgical conditions including sepsis, ischemia-reperfusion injury, and transplant rejection. Herbal products are being used as alternative treatments in such inflammatory conditions. Ginger is known for its ethno-botanical applications as an anti-inflammatory agent. 6-gingerol is one of the active ingredients of ginger that imparts ginger with its anti-inflammatory properties. We hypothesized that the anti-inflammatory effect of 6-gingerol is because of inhibition of macrophage activation, more specifically by an inhibition of pro-inflammatory cytokines and antigen presentation by lipopolysaccharide (LPS) activated macrophages. METHODS: To study the effect of 6-gingerol on pro-inflammatory cytokines, we measured the liberation of TNF-alpha, IL-1beta, and IL-12 by murine peritoneal macrophages exposed to several doses of 6-gingerol in the presence of LPS stimulation. We also studied the effect of 6-gingerol on the cell surface expression of B7.1, B7.2, and MHC II. Finally, we examined the APC function of the 6-gingerol treated macrophages by a primary mixed lymphocyte reaction. RESULTS: 6-gingerol inhibited the production of pro-inflammatory cytokines from LPS stimulated macrophages but had no effect on the LPS-induced expression of B7.1, B7.2, and MHC II. The APC function of LPS stimulated macrophages was also unaffected by 6-gingerol treatment. CONCLUSION: Our data indicate that 6-gingerol selectively inhibits production of pro-inflammatory cytokines from macrophages but does not affect either the APC function or cell surface expression of MHC II and costimulatory molecules. We, thus, provide a mechanistic insight into the anti-inflammatory properties of 6-gingerol that may be useful to treat inflammation without interfering with the antigen presenting function of macrophages.     

Diltiazem suppresses collagen synthesis and IL-1beta-induced TGF-beta1 production on human peritoneal mesothelial cells.

BACKGROUND: After long-term treatment with continuous ambulatory peritoneal dialysis (CAPD), some patients may develop peritoneal fibrosis. Peritoneal mesothelial cells (PMCs) participate in the inflammatory reactions in the peritoneal cavity, and transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are involved in peritoneal fibrosis. Diltiazem is used frequently in patients with CAPD to treat hypertension. The objectives of this study were to examine the effects of diltiazem on collagen- and IL-1beta-induced TGF-beta1 production on human PMCs and the signalling pathway of diltiazem in this induction. METHODS: Human PMCs were cultured from the enzymatic disaggregation of human omentum. Collagen synthesis was measured by [3H]proline incorporation into pepsin-resistant, salt-precipitated collagen. The expression of collagen I and III, and TGF-beta1 mRNA was evaluated by northern blotting. The production of TGF-beta1 by human PMCs was measured by immunoassay. The changes of intracellular calcium level after adding Fura-2-AM were measured by fluorescence spectrophotometry. Western blotting was used to assess mitogen-activated protein kinase (MAPK) signalling proteins. RESULTS: We found that diltiazem (<0.2 mM) inhibited collagen I and III mRNA expression and collagen syntheses on a dose-dependent basis. Diltiazem (0.2 mM) suppressed IL-1beta- (5 ng/ml) induced TGF-beta1 production on human PMCs at both the protein and mRNA levels. Diltiazem (0.2 mM) also inhibited IL-1beta- (5 ng/ml) induced collagen I and III mRNA expression. Intracellular calcium levels did not change after the treatment with diltiazem, IL-1beta or both. The IL-1beta-treated human PMCs increased phospho-JNK (stress-activated c-Jun N-terminal kinase) and phospho-p38 MAPK expression, while diltiazem could suppress this phenomenon. CONCLUSIONS: Diltiazem suppressed collagen synthesis of human PMCs and inhibited IL-1beta-induced TGF-beta1 production on human PMCs. This signalling transduction may be through p38 MAPK and JNK pathways instead of intracellular calcium. These results suggest diltiazem to be a potential therapeutic regimen in preventing peritoneal fibrosis and support further in vivo studies.     

In vitro effect of danazol on the cytokine production of peritoneal macrophages in patients with endometriosis

Recent evidence suggested that immunologic mechanisms might play a role in thepathogenesis of endo..t.io.is[1]. Women withendometriosis have an increased number andactivity of macrophages in peritoneal fluid(PF) and secretion of cytokines such as tumornecrosis factor (TNF ) and interleukin 6 (IL--6)by these cells.. In addition, autoantibodiessuch as antiendometrial antibody are often detected in peripherial blood samples obtainedfrom patients with endometriosis. Danazol, adrug commonly used…     

Inhibition of antigen-specific activation of an L3T4+ T cell line by cyclosporine with maintenance of macrophage-mediated antigen presentation

Cyclosporine has clearly been shown to directly inhibit T lymphocyte activation by monoclonal antibodies or mitogens where nominal antigen and accessory cells are not present. However, when T lymphocytes are stimulated by antigen, as occurs in allograft rejection, T lymphocytes and accessory cells must interact with one another. Under the latter circumstances, the issue of whether cyclosporine acts on T lymphocyte, accessory cell, or both is not resolved. This issue is addressed in this study. To assess the effect of cyclosporine on T cell activation, macrophages were incubated with heat-killed Listeria and then fixed in paraformaldehyde. These fixed macrophages retained their ability to present antigen to T cells but were not affected by subsequent treatment with cyclosporine. When cyclosporine and a L3T4+ T lymphocyte line were added simultaneously to fixed, antigen-pulsed macrophages, the drug inhibited antigen-specific T cell activation with a half maximal inhibitory concentration of 10 ng/ml. To our knowledge, this is the first evidence that low doses of cyclosporine inhibit antigen-specific T cell activation where the drug's effects on antigen-presenting cells have been excluded. To assess the effects of cyclosporine on macrophage-mediated antigen-presentation, macrophages were exposed simultaneously to cyclosporine and antigen, and then fixed. Antigen-presentation was not inhibited unless extremely large doses (9000 ng/ml) of cyclosporine were used. In our experimental system, any new inhibitory activity acquired by live cyclosporine-treated macrophages could be explained by residual drug. Finally, cyclosporine did not inhibit the induction of macrophage Ia expression nor antigen-presenting function after stimulation in vitro with lymphokine.     

Defective T-cell surface antigen expression after mitogen stimulation. An index of lymphocyte dysfunction after controlled murine injury.

Murine spleen T-cell activation in lectin-stimulated cultures after 25% body surface area burn injury or hind-limb amputation was studied by measuring the temporal expression of cell surface markers using monoclonal antibodies and two-color flow cytometry. Lymphocyte activation has been shown to be accompanied by the appearance of new surface antigens, including Interleukin-2 (IL-2) deceptor (IL-2R) and Ia, and emergence of cells that coexpress helper (Th) and suppressor (Ts) surface markers. IL-2R has been shown to appear early on stimulated cells, before DNA synthesis, whereas Ia appears later. Surface markers (L3T4, Lyt2, Ia, and IL-2R) were analyzed at time 0 and after 24, 48, and 72 hours of mitogen-stimulated culture. The appearance of IL-2R and Ia on Th (L3T4+) and Ts (Lyt-2+) populations was markedly depressed after burn injury, but minimal changes were seen after musculoskeletal injury. In addition, coexpression of L3T4/Lyt2 antigens was markedly reduced in burn-derived cells. Serum from burn-injured animals caused depression of surface antigen expression by stimulated normal cells. Recombinant IL-2, when added to burn-derived cell cultures, did not increase expression of these surface markers during culture, nor did it improve proliferation.     

Kinetics of interleukin-1 and tumor necrosis factor secretion by rabbit macrophages recovered from the peritoneal cavity after surgery

Macrophages play a crucial role in wound healing after surgical injury, both as scavenger cells responsible for wound debridement and as cells that secrete soluble factors such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). IL-1 and TNF alter many of the biological activities of cells that appear in postsurgical wounds. In this study, we determined the kinetics of IL-1 and TNF production by rabbit macrophages harvested from postsurgical peritoneal exudate (postsurgical macrophages) at several time points after peritoneal surgery. To further characterize the level of functional activities of postsurgical macrophages, the IL-1 and TNF levels were determined with or without stimulating the cells with lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). After surgery, the number of macrophages harvested by peritoneal lavage increased, reached peak levels on postsurgical day 3, and then decreased. IL-1 levels secreted by macrophages cultured without stimuli were elevated on postsurgical day 14 compared to the values on day 3 and 7. TNF concentrations peaked on days 1 and 14. In the conditioned culture media from LPS-PMA-stimulated macrophages, the levels of both IL-1 and TNF peaked on postsurgical days 3 and 14. These data suggest that the susceptibility of postsurgical macrophages to stimuli changes during the wound healing process with maximum sensitivity to the stimuli present during the early phase of peritoneal repair (day 3).     

Antigen processing and presentation by glomerular visceral epithelium in vitro

Although macrophages are considered the prototype of antigen presenting cells (APC), recent studies have emphasized the potential role of several parenchymal and mesenchymal cells in this process. We have studied the capacity of cultured glomerular visceral epithelial cells (GEC) to act as effective APC and compared this capacity with that demonstrated by peritoneal macrophages. Affinity-purified and in vitro propagated rat GEC were exposed to hen egg lysozyme, keyhole limpet hemocyanin, and cationic ferritin. As effector cells, we used antigen-specific T cell hybridomas; the level of antigen presentation was assessed by determining the level of interleukin 2 (IL-2) present in tissue culture supernatants. Cytokine-treated GEC were capable of processing and presenting all antigens in a dose-dependent manner. Crucial for antigen presentation were intracellular processing of antigen and the presence of Ia on the cell surface. Our findings indicate that GEC can act as effective APC, and further suggest that this capacity may be relevant to cell-mediated immune injury at the level of the glomerular capillaries in vivo.     

Mechanism of immunosuppression following hemorrhage: defective antigen presentation by macrophages

The mechanism by which simple hemorrhage profoundly impairs the proliferative response of T lymphocytes to mitogen and alloantigen, produces a defect in interleukin-2 generation, and increases the susceptibility to sepsis remains unknown. Since antigen presentation (AP) by the macrophage (M phi) plays a critical role in the antigen-specific activation of T-helper cells and lymphokine production, we investigated whether the function of the M phi as an AP cell is altered following hemorrhage. C3H/HEJ mice were bled to a mean BP of 35 mm Hg, maintained at that level for 1 hr, and then resuscitated. There was no mortality with this model. Control mice were not bled but otherwise treated identically. Immediately after resuscitation the mice were sacrificed and peritoneal M phi (PM phi) as well as splenic adherent cells (SAC) were harvested. AP function was tested by coculturing different numbers of PM phi and SAC with D10.G4.1 cells (2 x 10(4) cells/well) in the presence of conalbumin (300 micrograms/ml). This T-helper cell clone proliferates upon recognition of conalbumin in the context of Iak (a M phi surface membrane glycoprotein), thus directly reflecting M phi AP capability. After 72 hr of incubation, the cultures were pulsed with [3H]thymidine and harvested. D10.G4.1 proliferations induced via AP by PM phi and SAC from hemorrhaged-resuscitated mice were 29 and 24% of control, respectively (P less than 0.05). Thus, we conclude that AP by M phi following hemorrhage is defective despite adequate resuscitation, a mechanism which could explain the state of immunosuppression and enhanced susceptibility to sepsis.     

Do female sex steroids adversely or beneficially affect the depressed immune responses in males after trauma-hemorrhage?

HYPOTHESIS: Administration of female sex steroids in males after trauma-hemorrhage has salutary effects on the depressed immune responses. DESIGN: Randomized laboratory experiment. INTERVENTIONS: Male C3H/HeN mice were subjected to midline laparotomy and hemorrhagic shock (35+/-5 mm Hg for 90 minutes, then resuscitation) or sham operation and received subcutaneous 17beta-estradiol (40 microg/kg body weight) or corn oil vehicle at the beginning of resuscitation. MAIN OUTCOME MEASURES: At 24 hours after hemorrhage, the animals were killed and plasma 17beta-estradiol and IL-6, splenocyte interleukin (IL) 2, IL-3, and IL-10 production as well as splenic and peritoneal macrophage IL-1beta, IL-10, and IL-6 release were measured. RESULTS: Splenocyte IL-2 and IL-3 release were significantly depressed after hemorrhage in vehicle-treated mice (P<.05, analysis of variance). Treatment with 17beta-estradiol after hemorrhage led to the restoration of splenocyte IL-2 and IL-3 release. The depressed proinflammatory cytokine (IL-1 and IL-6) release seen in splenic and peritoneal macrophages was restored in the 17beta-estradiol-treated hemorrhage group. In contrast, the sustained release of the anti-inflammatory cytokine IL-10 by splenocytes and splenic and peritoneal macrophages in vehicle-treated mice after hemorrhage was decreased in 17beta-estradiol-treated mice. The increase in circulating IL-6 levels after hemorrhage was significantly attenuated in 17beta-estradiol-treated mice. Although administration of 17beta-estradiol increased plasma 17beta-estradiol levels by approximately 100% in sham as well as hemorrhage groups, improved immune responses were seen only in posthemorrhage 17beta-estradiol-treated mice. There was no adverse effect of 17beta-estradiol treatment in the sham or posthemorrhage groups. CONCLUSION: Since administration of a single dose of 17beta-estradiol in males after trauma-hemorrhage restores the immune functions and decreases circulating levels of IL-6, hormones with estrogenic properties should be considered as safe and novel therapeutic agents for restoring the immune responsiveness in male trauma victims.     

Potentiation of local lignocaine-induced sensory block by calcium channel blockers in rats

We have studied the effects of three different types of calcium channel blockers (verapamil, diltiazem, and nicardipine) on local lignocaine sensory block. The standardized tail flick test was used to measure the duration and degree of lignocaine-induced conduction block in rats. After obtaining baseline tail flick latencies (mean 3.2 s), two 100- microliter doses of 0.3% lignocaine alone, a combination of verapamil 25, 100 or 200 micrograms, diltiazem 25, 100 or 200 micrograms, or nicardipine 0.5, 1.0 or 2.0 micrograms, and a large dose of calcium channel blockers (verapamil 200 micrograms, diltiazem 200 micrograms or nicardipine 2.0 micrograms) were injected on opposite sites of the tail base and the tail flick test was performed every 5 min for 45 min. A large dose of the calcium channel blockers showed no prolongation of tail flick latencies. Administration of 0.3% lignocaine alone produced a significant increase in tail flick thresholds and the peak effect of the percentage maximum possible effect (% MPE) was demonstrated at 5 min after drug injection (mean % MPE 28.8%; P < 0.01 vs baseline). Co- administration of 0.3% lignocaine and three doses of verapamil produced significant increases in area under the curve (AUC) in a dose-dependent fashion. Mean AUC values for 0.3% lignocaine alone and a combination of verapamil 25, 100 or 200 micrograms were 217.5, 502.5, 529.1 and 1600.3, respectively. Almost similar patterns of augmentation in AUC values were demonstrated after addition of different doses of diltiazem or nicardipine to 0.3% lignocaine. We conclude that the use of mixtures of local anaesthetic and calcium channel blocker potentiated lignocaine sensory block at the level of the peripheral nerves.      

Beneficial effect of enhanced macrophage function in the trauma patient.

Host immunosuppression after trauma contributes to septic morbidity. The macrophage is a key element in the host immune response. This study evaluated glucan, a macrophage stimulant, in a prospective, randomized, double-blind study of 38 trauma patients undergoing surgery. Glucan (21 patients), 50 mg/m2, or placebo (17 patients) was given intravenously daily for 7 days. Delayed hypersensitivity skin testing was performed on days 1 and 7 after trauma. Serum interleukin-1 (IL-1) and tumor necrosis factor (TNF) were assayed after trauma. While the total mortality rate was significantly less in the glucan group (0% versus 29%) (p less than 0.05), the mortality rate from sepsis was not statistically different (0% versus 17.6%). Glucan therapy significantly decreased septic morbidity (9.5% versus 49%; p less than 0.05). Serum IL-1 had a greater increase in glucan patients on day 3 after trauma (143.4 +/- 19.3% versus 78.6 +/- 11.7%; p less than 0.05), but there was no difference thereafter. Serum TNF did not vary between groups. Early increase in IL-1 correlated with subsequent skin test conversion to positive. Neither serum IL-1 nor TNF was a reliable indicator of future sepsis. Further clinical trials are indicated to evaluate biologic response modifiers that activate macrophages in the trauma patient.