A cyclophosphamide-sensitive suppressor T cell circuit induced by thermal injury

The immunoregulatory events leading to the generation of suppressor T cells after burn injury were studied by means of a mouse burn model. Immunoresponsiveness was assessed on various postburn days with a primary in vitro mixed lymphocyte reaction (MLR). Responses of burned mice were depressed between 3 and 14 days after burn. Suppression became significant 5 days after burn and remained so through 14 days after burn. The Ly-t surface phenotype of T cells was studied on days 5 and 7 after burn. On day 5 burn, T cells responsible for suppression bore an Ly-1+, 2- phenotype. By postburn day 7 suppressor T cells bore an Ly-1-,2+ phenotype. These observations are consistent with the notion of feedback suppression after burn injury, wherein Ly-1+, 2- T cells induce a subset of Ly-1+,2+ T cells, thereby generating Ly-1-,2+ suppressor effector T cells. An attempt was made to uncouple the putative suppressor inducer and the suppressor effector using low doses of cyclophosphamide (CY). A single dose of 25 mg CY/kg resulted in transient restoration of depressed burn responses. By using multiple sequential doses of CY, this restorative effect could be prolonged through 8 days after burn. A dosage schedule that restores responses of burn cells without affecting responses of control cells is presented.     

Suppressor T-cell levels are unreliable indicators of the impaired immune response following thermal injury.

The presence of increased levels of suppressor T cells after thermal injury and their relevance remain controversial. It is unclear whether suppressor T cells are the cause or result of sepsis complicating thermal injury. Spleen cells from a standardized murine burn model and sham burn controls were studied and the relationship between the levels of suppressor cytotoxic T cells (CD8, Lyt-2+), helper T cells (CD4, L3T4+), response to concanavalin A (ConA) and to phytohemagglutinin (PHA) and interleukin-2 (IL-2) production was examined. Mortality following infection via cecal ligation and puncture (CLP) of matched controls was also studied. At day 7 postburn, mean ConA (70 +/- 12% of control) and PHA response (58% +/- 5.2% of controls) and IL-2 production (43% +/- 5.4%) were significantly less than sham burn values (100%; p less than 0.05). However, the mean percentage of cells staining with anti-Lyt-2 and anti-L3T4 (9.1 +/- 0.59 and 13.9 +/- 0.65) was similar to the mean percentage in sham burn animals (9.4 +/- 0.65 and 16.6 +/- 1.1). Furthermore, no significant differences were observed between burned mice and controls in helper (17.3% +/- 1.8% burn vs. 21.2% +/- 1.7% sham) or suppressor cell levels (7.8% +/- 1.2% burn vs. 8.6% +/- 0.7% sham) or helper-suppressor ratios on day 10 postburn. Mortality of 20 litter-matched controls subjected to CLP on day 10 postburn was 90%, which was significantly greater than the sham burn mortality of 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major injury induces increased production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection.

OBJECTIVE: The purpose of this study was to compare the production of interleukin-10 (IL-10) by peripheral blood mononuclear cells (PBMC) from injured patients and control subjects to determine the responsible cell types and to relate IL-10 production to the occurrence of sepsis. A mouse model of burn injury was used to confirm the human findings and to assess the importance of IL-10 in the lowered resistance to infection after injury. SUMMARY BACKGROUND DATA: Severe injury is associated with depressed immune responses. Although IL-10 is known to inhibit several aspects of immune reactivity, the role of IL-10 in postinjury immune suppression remains controversial. METHODS: Peripheral blood mononuclear cells from 14 burn and 12 trauma patients and 16 healthy individuals were studied at serial intervals for IL-10 production stimulated by a T-cell mitogen, phytohemagglutinin, and by bacterial lipopolysaccharide. To determine the source of IL-10, CD4+ and CD8+ lymphocyte subsets were obtained by selective depletion of PBMC with antibody-coated magnetic beads and were stimulated by anti-CD3 antibody to induce IL-10 secretion. In addition, IL-10 production by patients' PBMC in the first 10 days after injury was assessed for correlation with subsequent septic events. Anti-CD3-stimulated IL-10 production also was determined for CD4- and CD8-enriched lymphocyte subsets obtained by antibody and complement depletion of splenocytes harvested from groups of burn and sham burn mice at day 10 after injury, the time of maximal susceptibility to a septic challenge, cecal ligation and puncture (CLP). Finally, to test the importance of IL-10 in immune suppression in vivo, groups of burn and sham burn mice were treated with anti-IL-10 monoclonal antibody or control immunoglobulin G (IgG) on days 1 and 3 postinjury and were observed for survival after CLP on day 10. RESULTS: Patients' PBMC produced significantly more IL-10 than did controls' PBMC 7 to 14 days after injury. Patients' CD4+ (T-helper) but not CD8+ (T-cytotoxic) lymphocytes also showed increased IL-10 production versus those of control subjects early after injury. Increased PBMC IL-10 production in the first 10 days postinjury correlated significantly (p < 0.05) with subsequent septic events. Burn mouse CD4-enriched but not CD8-enriched splenocytes produced more IL-10 than did sham burn splenocyte subsets on day 10 after injury. Burn mice treated with anti-IL-10 antibody but not with control IgG had significantly increased survival after CLP. CONCLUSION: Serious injury in humans and in a mouse burn model is followed by increased stimulated production of IL-10 by cells of the immune system. The CD4+ T-helper cells appear to be a major source of IL-10 after injury. In injured patients, increased IL-10 production is correlated with subsequent septic events, and in the burn mouse, IL-10 appears to induce decreased resistance to infection.     

Injury induces deficient interleukin-12 production, but interleukin-12 therapy after injury restores resistance to infection

OBJECTIVE: To assess at serial intervals the production of interleukin-12 (IL-12) by monocytes/macrophages from the peripheral blood of injured patients and control subjects, and using a mouse model to confirm human findings and explore the effectiveness of low-dose IL-12 therapy in restoring resistance to infection after injury. SUMMARY BACKGROUND DATA: Serious injury is associated with loss of function of the T helper 1 lymphocyte phenotype, but little is known about IL-12 production in injured patients. The authors previously reported that early, moderate-dose IL-12 therapy in a mouse model of burn injury restored resistance to a later infectious challenge (cecal ligation and puncture, CLP). However, the efficacy of clinically relevant low-dose IL-12 therapy carried out to or beyond the time of septic challenge remains to be tested. METHODS: Peripheral blood mononuclear cells (PBMCs) and adherent cells were obtained from 27 patients with major burns or traumatic injury and 18 healthy persons and were studied at serial intervals for IL-12 production stimulated by bacterial lipopolysacharide (LPS). PBMCs from 18 of the same patients were studied for IL-10 production as well. IL-12 production by adherent cells from the spleens of burn or sham burn mice was studied at serial intervals after injury to confirm the human findings. Low-dose IL-12 or vehicle was given every other day to groups of burn and sham burn mice, which were then challenged with CLP on day 10, and survival was determined. Finally, spleens were harvested from burn or sham burn animals receiving low-dose IL-12 or vehicle after CLP. After splenic cellularity was determined by hemocytometer, splenocytes were cultured and production of tumor necrosis factor-alpha, interferon-gamma, and IL-10 were assessed by immunoassay. RESULTS: Adherent cells from patients' PBMCs produced significantly less IL-12 than normal PBMCs after injury, reaching a nadir 8 to 14 days after injury. Stimulation of whole PBMCs by LPS indicated that at 8 to 14 days after injury, IL-12 production by PBMCs was significantly lower and IL-10 production was significantly higher than that of PBMCs from healthy persons. Low-dose IL-12 therapy significantly increased survival after CLP. Splenocytes from burn mice treated with IL-12 had significantly increased production of TNF-alpha and IF-beta, both before and after CLP, when compared with vehicle-treated burn animals. IL-10 production by bum splenocytes remained high after IL-12 treatment. Splenic cellularity increased after IL-12 treatment in burn mice. CONCLUSION: The capacity to produce IL-12 by adherent cells of the monocyte/macrophage lineage is significantly reduced after serious injury in humans and in a mouse burn model. In humans, there is a reciprocal relation between diminished IL-12 production and increased IL-10 production at approximately 1 week after injury. Low-dose IL-12 therapy in the mouse burn model markedly increased survival after a septic challenge, even when treatment was carried beyond the onset of sepsis. Low-dose IL-12 treatment in the mouse increased production of proinflammatory mediators important in host defense and at the same time maintained or increased production of IL-10, an important antiinflammatory cytokine.     

Effect of protein malnutrition and immunomodulation on immune cell populations.

Malnutrition has deleterious effects on immune functions, which predispose to an increased risk of infection. To study the effect of protein malnutrition on such immune functions and resistance to infection, we divided C57BL/6 mice into three groups: (1) control-standard diet, (2) protein-malnourished for 14 days, and (3) protein-malnourished for 14 days followed by standard diet for 3 days. The animals were further divided into subgroups: (1) an untreated group, (2) a muramyl dipeptide (MDP)-treated group, and (3) an interferon-gamma (IFN-gamma)-treated group before cecal ligation and puncture (CLP). Malnourished mice had significantly (P less than 0.05) lower body weight, serum albumin, spleen/body weight, percentage of splenic macrophages with Ia expression (%MOIa), increased splenic T suppressor cells, and greater mortality after CLP. Refeeding plus IFN-gamma or MDP significantly increased %MOIa (P less than 0.05) and also abrogated the increase in splenic lymphocytes seen in the malnourished animals. The increase in splenic suppressor T cells was not affected by refeeding or immunomodulation. Mortality after CLP was increased from 15% in the controls to 85% in the malnourished group and was significantly decreased by refeeding, MDP, and the combination of refeeding plus immunomodulators (P less than 0.05). These data show that 14-day malnutrition adversely affected the immune response to infection and increased mortality from CLP. Refeeding and immunomodulation restored macrophage Ia expression without CLP but not after the procedure, despite the significant reduction in mortality. The use of immunomodulation in protein malnourished conditions may serve as an adjuvant role to nutritional support.     

Inhibition of CD1d activation suppresses septic mortality: a role for NK-T cells in septic immune dysfunction

BACKGROUND: Studies indicate that following septic insult there is development of generalized immune dysfunction in T cells, B cells and phagocytes, which is thought to contribute to morbidity and mortality. Specifically, there is a shift in the lymphocytes of septic animals toward an increased release of Th2 cytokines. NK-T cells have been shown to contribute to propagation of the Th2 response. The influence of NK-T cells on the immune response to septic challenge is poorly understood. In this study, we examine whether NK-T cells contribute to the immune dysfunction seen following the onset of polymicrobial sepsis, as produced by cecal ligation and puncture (CLP). MATERIALS AND METHODS: Male 129S1/SvImJ mice were pretreated with either rat IgG (isotypic control) or monoclonal antibody to CD1d (clone 1B1) (0.5 mg), which blocks signaling/antigen presentation via the CD1d cell surface receptor, thereby, ablating the activation and differentiation of the NK-T cells. Septic survival with and without anti-CD1d (CLP/CD1d) pretreatment was assessed. Mice sacrificed 24 h after CLP were assessed for change in splenic %NK-T cell (via flourescense activated cell sector) and for splenic, hepatic, and lymphoid/macrophage production of pro-inflammatory or anti-inflammatory cytokines (via enzyme-linked immunosorbent assay). RESULTS: Administration of anti-CD1d reduced septic mortality 35% at 6-10 d (n = 23 mice/group) (P <.05). There was a consistent increase in the %CD3(+) NK1.1(+) cell population (NK-T cells) in septic mice (1.706%), which was markedly suppressed by pretreatment with anti-CD1d (0.592%). IL-6 and IL-10 levels were suppressed by anti-CD1d in the spleen and blood. CONCLUSIONS: Together these findings imply not only that NK-T cells may play a role in mediating the immune suppression seen in bacterial sepsis, but that inhibition of their activation promotes survival to septic challenge.     

T suppressor cells induced by transfusions and cyclosporine. Studies in the murine cardiac allograft model

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.     

Immunomodulating drugs increase resistance against sepsis in traumatized mice

Immune suppression occurs frequently after major injury and undoubtedly contributes to infection and mortality in trauma patients. Prevention of such suppression may lead to decreased infection and improved survival in trauma patients surviving the immediate insult of injury. Suppressor-cell activation appears to play a key role in immune suppression after major injury. For several years we have studied the effects on immune functions after injury of various drugs which have been shown in the immunologic literature to have inhibitory effects on suppressor cell populations. H2-antagonists may inhibit suppressor cell activation by blocking surface H2-receptors, which are present in higher numbers on suppressor cells than on helper cells. Prostaglandin inhibitors may block the multiple immune suppressive effects of prostaglandins, particularly PGE2. Immunological studies suggest that low-dose cyclophosphamide selectively inhibits the proliferation of suppressor T cells. Our previous work suggested that such drugs preserve cell-mediated immune functions after injury. In experiments reported here, we utilized a standard hindlimb crush injury and amputation in mice, followed in 24 hrs by polymicrobial septic challenge using cecal ligation and 23-gauge needle puncture (CLP). Nontraumatized (control) mice had a 36.2% mortality after CLP; when crush injury/amputation was followed by CLP in 24 hrs the mortality rose to 63.8% (p less than 0.0035). When mice were given 24 hrs before crush/amputation: cimetidine, an H2-antagonist (10 mg/kg/day); ibuprofen, a prostaglandin blocker (5 mg/kg/day); or cyclophosphamide (2.5 mg/kg/day), and a second dose on the day of trauma, with CLP 24 hrs later, subsequent mortality was not different from the mortality in nontraumatized, control mice (p less than 0.0035).(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressor cell numbers and activity in non-transfused renal dialysis patients

Suppressor cell activity was studied in 30-non-transfused renal dialysis patients using the Concanavalin A enhancement method. Suppressor cell numbers were estimated in 19 patients by the T suppressor (T gamma) rosette technique and in 15 patients using a monoclonal antibody (OKT8) specific for T suppressor cells. The role of prostaglandin secreting monocytes in the observed suppression was investigated in 6 patients using the prostaglandin secreting inhibitor flurbiprofen. Both suppressor cell numbers and activity were either normal or low compared with normal healthy controls. Flurbiprofen at concentrations of 10(-4) and 10(-6) M caused a decrease in activity in both patients and normal controls. The results indicate that the increased suppressor cell activity and number reported in dialysis patients may be due to the effect of blood transfusion previously administered to the patients.     

Mechanism of immune dysfunction in sepsis: inducible nitric oxide-meditated alterations in p38 MAPK activation

BACKGROUND: After the onset of sepsis, there is a marked dysfunction in cell-mediated immunity that contributes to the morbidity and mortality seen in this condition. Although both nitric oxide (NO) from inducible NO synthase (iNOS) and the activation of p38 mitogen-activated protein kinase (p38 MAPK) appear to contribute to this immune dysfunction, the extent to which NO regulates p38 MAPK activity in sepsis remains unknown. METHODS: To examine this, we induced sepsis by cecal ligation and puncture (CLP) in iNOS knockout (iNOS -/-) or C57BL/6 control mice. Twenty-four hours after CLP or sham operation, splenic T cells and macrophages were isolated and then stimulated with monoclonal antibody against the T-cell marker CD3 (anti-CD3) or lipopolysaccharide. At 4 or 24 hours after stimulation, cytokine release was determined by enzyme-linked immunosorbent assay, and p38 MAPK phosphorylation (activation) was determined by immunoblotting with antibody specific to phosphorylated p38 MAPK. RESULTS: Splenic T-cell p38 MAPK activation and interleukin (IL)-10 release was increased by CLP, whereas Th1 cytokine (IL-2, interferon-gamma) release was depressed. iNOS gene deficiency inhibited p38 MAPK activation in splenic T cells taken from septic mice, and also suppressed IL-10 release in both sham and septic mice. Interestingly, although deficiency of iNOS restored IL-2 release after CLP, both sham and CLP T cells remained depressed in their ability to release interferon-gamma. Septic insult markedly suppressed C57BL/6 splenic macrophage release of proinflammatory agents tumor necrosis factor, IL-12, and IL-1, while augmenting the release of IL-10. However, although deficiency of iNOS concomitantly restored the ability to produce tumor necrosis factor while suppressing the rise in IL-10 release and p38 MAPK activation, it only partially restored IL-1 release and had no effect on IL-12 production seen after CLP. CONCLUSION: These data suggest that NO release from iNOS regulates aspects of sepsis-induced immune dysfunction by the activation of p38 MAPK.     

Interleukin-12 and -18 induce severe liver injury in mice recovered from peritonitis after sublethal endotoxin challenge

BACKGROUND: Postoperative intraabdominal abscess is the major complication after abdominal surgery, and additional infection is often observed and becomes the leading cause of death in septic patients who survive initial resuscitation. Sepsis is initiated and perpetuated by the overzealous systemic production of proinflammatory cytokines-such as tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-12, and IL-18-sometimes resulting in excessive tissue injury and death. The purpose of this study was to assess the correlation between liver and spleen innate cytokine responses and organ dysfunction in sepsis syndrome. METHODS: Peritonitis was induced by cecal ligation and puncture (CLP). All CLP mice survived more than 7 days after the procedure, and serum cytokine (TNF-alpha, IL-12, IL-18, and IL-10) levels peaked 12 hours after CLP; thereafter, they returned to basal levels 7 days after CLP. The mice were injected with a sublethal dose of lipopolysaccharide (LPS) 7 days after CLP. Survival rates, tissue damage, serum cytokine levels, and cytokine production of liver or spleen mononuclear cells (MNCs) were evaluated. RESULTS: All CLP mice died within 6 hours from liver injury 7 days after LPS challenge, but all sham mice survived. IL-12, IL-18, and IFN-gamma levels in supernatants of the liver MNCs stimulated with LPS in CLP mice were significantly higher than those in sham mice 7 days after the procedure. Furthermore, serum IL-12 and IL-18 levels and liver MNCs IL-12, IL-18, and IFN-gamma production were significantly increased in CLP mice compared with sham mice after LPS challenge. Thereafter, effects of anti-IL-12 and/or anti-IL-18 antibody were evaluated in LPS-injected CLP mice. The survival rate of LPS-injected CLP mice treated with both anti-IL-12 and anti-IL-18 antibody was significantly better than that of untreated mice. Furthermore, liver damage was improved. CONCLUSION: Mice recovered from mild peritonitis died of severe liver injury by subsequent injection of a sublethal dose of LPS, and this liver injury was related to the collaborating production of IL-12 and IL-18 by liver MNCs.     

The effect of in vivo T helper and T suppressor lymphocyte depletion on wound healing.

The role of T lymphocytes in wound healing is still not well-defined. Because it had been previously shown that in vivo depletion of T cells leads to impaired wound healing, the effect of depleting T cell subsets on subsequent fibroplasia was studied. T helper/effector cells were depleted by the use of the monoclonal antibody GK1.5, reactive against the L3T4 antigen (CD4). T suppressor/cytotoxic lymphocytes were depleted by using the 2.43 monoclonal antibody reactive against the Lyt 2 antigen (CD8). In the first experiment, Balb/c mice were treated with the antibodies starting at 24 hours before wounding was performed, and weekly thereafter. Depletion of the T helper/effector cells had no effect on wound-breaking strength or hydroxyproline deposition in sponge granulomas, whereas depletion of T suppressor/cytotoxic cells significantly enhanced both of these healing parameters. In a second experiment, T cell subset depletion was started on Days 0, 3, 7, 10, and 14 postwounding, and treatments were continued weekly thereafter. Once again, depletion of T helper/effector cells had no effect on wound healing, whereas depletion of T suppressor/cytotoxic cells markedly increased both wound-breaking strength and collagen synthesis. In conclusion, the data show that T suppressor/cytotoxic cells have a counter-regulatory role in wound healing, whereas the T cell subset responsible for up-regulating wound healing remains to be identified.     

Captopril and immune regulation

We examined the in vitro effect of captopril (2.5 to 5 micrograms/ml) on the primary antibody response of human B cells. Captopril suppresses (by 50%) the specific anti-trinitrophenyl (TNP) response of unfractionated peripheral blood mononuclear cells (PBM) but not that of nonadherent PBM. The susceptibility to captopril suppression can be restored in the latter cell cultures by 10% adherent radioresistant cells. This suppression is independent of prostaglandins. In transfer experiments, cells preincubated with 5 micrograms/ml captopril suppress the antibody response of autologous nonadherent PBM. The inductive phase of this suppression requires both adherent cells and radiosensitive T cells. Once induced, the suppression can be transferred by isolated T effector cells. In vivo after a unique oral intake of captopril a moderate suppressor activity can be demonstrated in adherent cells from normal individuals. We conclude that captopril interferes with the immune regulation by inducing a suppressor circuit involving monocytes and a T8 suppressor effector lymphocyte.     

Defective antigen presentation to a cloned T helper cell by macrophages from burned mice can be restored with interleukin-1

T helper (Th) cell dysfunction occurring very early (i.e., 24 to 72 hours) after a 30% full-thickness burn in a murine model cannot be attributed to suppressor T cell activity. Th cell activity is influenced by the activity of antigen-presenting cells (APCs). These cells process antigen and present a complex of antigen and cell surface Ia to the T cell. Additionally, they elaborate interleukin-1 (Il-1), and these events lead to Th cell release of Il-2, expression of Il-2 receptors, and proliferation of Th cells. We examined the contribution of APCs to postburn Th cell dysfunction by using mitomycin C-treated spleen cells from normal and burned mice as an APC population. The Th cell population consisted of a cloned Th cell line (D10.G4.1) that recognizes conalbumin in the context of I-Ak and proliferates when approximately stimulated. We found that APCs from burned mice induced significantly less Th cell proliferation (p less than 0.05). This was true of unfractionated spleen cells (50.4% of control) as well as positively selected (44.2% of control) or negatively selected (51.9% of control) splenic APCs. When cocultured with APCs from control mice, APCs from burned mice did not suppress control values of Th cell proliferation. Finally, the addition of murine Il-1 in vitro to cultures of burn-derived APCs, antigen, and T cell clone restored Th cell proliferation to control levels (from 38.3% to 92.8%) without nonspecifically enhancing similar cultures employing normal APCs. Il-1 in vitro did not improve Th cell function in the absence of antigen. Thus splenic APCs from mice exhibit defective antigen presentation early after burn injury. This defect is not a result of suppressor factor production by burn APCs and can be restored by Il-1 in vitro. Th cell dysfunction early after burn injury is thus due, in part, to APC dysfunction.     

Is circulating endotoxin the trigger for the systemic inflammatory response syndrome seen after injury?

OBJECTIVE: Patients with severe traumatic or burn injury and a mouse model of burn injury were studied early after injury to determine the relation of plasma endotoxin (lipopolysaccharide [LPS]) to the production of proinflammatory cytokines and subsequent resistance to infection. SUMMARY BACKGROUND DATA: Elevated levels of plasma LPS have been reported in patients after serious injury. It has been suggested that circulating LPS may be a trigger for increased proinflammatory cytokine production and may play a role in the septic syndromes seen in a substantial portion of such patients. Yet, despite multiple reports of leakage of LPS from the gut and bacterial translocation after injury in animal models, there is little direct evidence linking circulating LPS with production of inflammatory mediators. METHODS: The authors studied serial samples of peripheral blood from 10 patients with 25% to 50% surface area burns and 8 trauma patients (injury Severity Score, 25-57). Patients were compared with 18 healthy volunteers. The study was focused on the first 10 days after injury before the onset of sepsis or the systemic inflammatory response syndrome. Plasma samples were assayed for LPS, and adherent cells from the blood were studied for basal and LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6). The correlation of increased plasma LPS with TNF-alpha production was studied as was the association of increased plasma LPS and increased TNF-alpha production with subsequent septic complications. We also studied a mouse model of 25% burn injury. Burn mice were compared with sham burn control subjects. Plasma samples were assayed at serial intervals for LPS, and adherent cells from the spleens were studied for basal- and LPS-stimulated production of TNF-alpha, IL-1 beta, and IL-6. Expression of the messenger RNAs for IL-1 beta and TNF-alpha also was measured. The relation of increased TNF-alpha production with mortality from a septic challenge, cecal ligation and puncture (CLP), was determined. Finally, the effect of administration of LPS to normal mice on subsequent mortality after CLP and on TNF-alpha production was studied. RESULTS: Elevated plasma LPS (> 1 pg/mL) was seen in 11 of the 18 patients within 10 days of injury and in no normal control subjects. In this period, patients as compared with control subjects showed increased stimulated production of TNF-alpha, IL-1 beta, and IL-6. Increased TNF-alpha production was not correlated with elevated plasma LPS in the same patients. Neither increased plasma LPS nor increased TNF-alpha production early after injury was correlated with subsequent development of systemic inflammatory response syndrome or sepsis in the patients. Burn mice, as compared with sham burn control subjects, showed elevated plasma LPS levels chiefly in the first 3 days after injury. Increased stimulated production of proinflammatory cytokines by adherent splenocytes from the burn mice also was seen at multiple intervals after injury and did not correlate with mortality from CLP. Increased production of TNF-alpha and IL-1 beta was associated with increased expression of messenger RNAs for these cytokines. Finally, two doses of 1 ng LPS administered 24 hours apart to normal mice had no effect on mortality from CLP performed 7 days later nor on the production of TNF-alpha at the time of CLP. CONCLUSIONS: These findings call into question the idea that circulating LPS is the trigger for increased proinflammatory cytokine production, systemic inflammatory response syndrome, and septic complications in injured patients.     

Induction of transplantation tolerance in rats by spleen allografts. I. Evidence that rats tolerant of spleen allografts contain two phenotypically distinct T suppressor cells

We have examined suppressor cell activity in transplantation tolerant (TT) rats bearing vascularized spleen allografts in several different donor-recipient combinations. More than 60% of WAG (RT-1u) and 65% of AGUS (RT-1l) spleen allografts were permanently accepted when transplanted to AGUS and PVG (RT-1c) rats, respectively. All (WAG X AGUS)F1 to AGUS and (AGUS X PVG)F1 to PVG spleen allografts survived indefinitely. Unseparated LNC, TDL, and whole T cell or W3/25+, OX8- T cell populations obtained from AGUS rats bearing (WAG X AGUS)F1 spleens exhibited reduced mixed lymphocyte reaction (MLR) responses to the spleen donor, and to some extent to BN(RT1n) third-party stimulators, but responded normally to PVG.A(RT1a) stimulators. Coculture experiments demonstrated that lymph node cells (LNC) and thoracic duct lymphocytes (TDL) of TT rats contain RT1 specific suppressor cells. Furthermore, T cells isolated from all donor-recipient combinations contained two phenotypically distinct suppressor cell populations: a radiosensitive W3/25+, OX8- (Th/i) and a relatively radioresistant W3/25-, OX8+ (Ts/c). These Ts may be responsible for the maintenance of TT.     

Regulation of delayed-type hypersensitivity. VI. Antigen-specific suppressor T cells and suppressor factor for delayed-type hypersensitivity to histocompatibility antigens

Mice develop highly significant levels of delayed-type hypersensitivity (DTH) to major and minor histocompatibility antigens when injected s.c. with lymphoid cells from X-irradiated allogeneic donors. However, when mice are inoculated i.v. with a high dose of X-irradiated allogeneic lymphoid cells, they not only fail to develop DTH to the allogeneic cells, but their ability to respond to an immunogenic challenge of the alloantigens is also significantly depressed. This suppression is adoptively transferable by antigen-specific suppressor T cells and not by immune serum. Cell surface phenotypic analysis shows that the primary suppressor cells for alloantigens are Thy-1+, Lyt-1+2-, and Ia-, whereas the secondary suppressor cells appearing after boosting injection are Thy-1+, Lyt-1+2+, and Ia-. These suppressor T (Ts) cells localize in the lymphoid organs shortly after their induction and are largely absent from the spleen or lymph node 1 month later. However, "suppressor memory" can be recalled by an immunogenic dose of alloantigens which would normally induce DTH effector cells rather than suppressor cells in naive mice. When the suppressor cells were cultured in vitro for 48 hr, the supernatant contained suppressive activity. It appears likely that the manifestation of the suppressor cells is via soluble, antigen-specific suppressor factor(s), the production of which is dependent on viable T cells.     

Temporal correlation of impaired immune response after thermal injury with susceptibility to infection in a murine model

Suppression of cellular immunity and increased susceptibility to sepsis frequently accompany thermal injury. However, a convincing association between the two has been difficult to establish in human beings. Therefore we chose to investigate the relationship of impaired cell-mediated immunity with susceptibility to sepsis in an animal model. We studied the response to phytohemagglutinin (PHA) and interleukin-2 (IL-2) production by splenocytes from mice subjected to a standard 25% scald burn and killed at intervals of 3, 5, 7, 10, 14, and 25 days after thermal injury. Burned mice were compared in all instances to sham-burn animals (i.e., animals that had been anesthetized and shaved but not burned). We also studied mortality after cecal ligation and puncture (CLP), as a septic challenge, in burned and control animals at the same postburn intervals. We found maximal suppression (50% to 55%) of the PHA response at 10 to 14 days after injury and maximum suppression (68%) of IL-2 production at 7 days. Both of these parameters returned to normal by postinjury day 28. Mortality after CLP increased gradually from control levels after thermal injury up to a maximum of 88% on postburn day 10 and also returned to control levels after 28 days after burn. Significant correlations were found between mortality after CLP in the postburn period and suppression of the PHA response, on the one hand, and the suppression of IL-2 production, on the other (r = 0.89 and 0.91, respectively; p less than 0.05). This result implies a causal relationship between impaired cell-mediated immunity and susceptibility to sepsis after burn injury.