Exogenous IL-12 and its effect on TH1/TH2 cell activity after cardiac surgery.

There is controversy about the origin of the alterations in T helper 1 (TH1)/TH2 cell activity after major surgical procedures such as on-pump cardiac surgery. We hypothesized that a postoperative decrease in interferon (IFN) gamma-producing TH1 lymphocyte activity may be the sole cause of this TH1/TH2 shift and that the addition of recombinant IL-12 can reverse TH1 suppression. Peripheral blood mononuclear cell fractions from 20 low-risk elective cardiac surgery patients were analyzed preoperatively (d0) and on the first (d1), third (d3), and sixth (d6) postoperative days. We determined the absolute numbers of T helper lymphocytes, IFN-gamma-producing TH1 cells, and IL-4-producing TH2 cells after stimulation and measured IFN-gamma and IL-4 levels in the supernatants of stimulated peripheral blood mononuclear cell cultures, absolute monocyte counts, human leukocyte antigen-DR expression, and intracellular IL-12 synthesis under comparable conditions. Recombinant IL-12 alone or in combination with a neutralizing antibody was added. T helper lymphocyte counts were reduced postoperatively from d1 to d6 (P < 0.05). Absolute IFN-gamma- and IL-4-positive T helper lymphocyte counts were reduced on d1 (P < 0.05). Intracellular IL-4 production in T helper lymphocytes remained postoperatively unchanged. Interferon gamma synthesis was significantly reduced until d3 (P = 0.001) and significantly increased after IL-12 addition (P < 0.05). This effect was reversed by the addition of a neutralizing anti-IL-12 antibody. The TH1/TH2 shift after cardiac surgery seems to be caused primarily by a decrease in cellular IFN-gamma synthesis in TH1 lymphocytes. Because TH1 suppression can be reversed by IL-12, it is more likely to be the result of altered stimulation pathways than cellular defects.

Postoperative PMBC-derived IL-6 and TNF-alpha-release is uninfluenced by IL-12-mediated restoration of IFN-gamma synthesis.

OBJECTIVES: Interferon gamma (IFN-gamma) synthesis in peripheral blood mononuclear cells (PBMCs) is suppressed after major surgical trauma. Interleukin-12 (IL-12) has been shown to stimulate IFN-gamma-synthesis. We hypothesised that exogenous IL-12 can increase perioperative pro-inflammatory cytokine release. We therefore assessed the effect of IL-12 on IFN-gamma-synthesis and pro-inflammatory cytokine release in vitro before and after cardiac surgery. DESIGN: In this prospective study, PBMCs from 20 elective cardiac surgery patients were stimulated for 24 hours with staphylococcal enterotoxin B and lipopolysaccharide before surgery (d0) and on the 1st (d1), 3rd (d3) and 5th (d5) postoperative days. IL-12 was added at each time point investigated. IFN-gamma, IL-6, tumour necrosis factor-alpha (TNF-alpha), IL-2, IL-4, IL-5, and IL-10 concentrations were assayed. RESULTS: IFN-gamma-synthesis was significantly reduced at d1, d3 and d5. When IL-12 was added, IFN-gamma-synthesis returned to preoperative levels at d1, d3 and d5. Neither IL-6 nor TNF-alpha-synthesis was influenced by IL-12. CONCLUSIONS: IFN-gamma synthesis is significantly reduced after major surgical trauma. IL-12 increases IFN-gamma-synthesis before and after surgery without influencing pro-inflammatory cytokine synthesis.

A comparison of monocyte counts and ex vivo and in vitro monocyte cytokine production after major surgical trauma.

BACKGROUND: Impaired function of cluster of differentiation 14-positive (CD14+) monocytes (MOs) after major surgical trauma is believed to predispose to infectious complications. Postoperative decreases in human leukocyte antigen (HLA)-DR expression, tumor necrosis factor-alpha (TNF-alpha) production and interleukin (IL)-12 synthesis have been reported. There are no studies comparing absolute MO counts and MO cytokine synthesis in peripheral blood and stimulated cultures. METHODS: The study group included 10 low-risk patients undergoing elective cardiac surgery with extracorporeal circulation. Preoperatively (d0) and on the first (d1) and third (d3) postoperative d, we analyzed leukocyte counts, CD14+ MO absolute counts, HLA-DR expression, and stimulated IL-12 and TNF-alpha synthesis using flow cytometry. In addition, IL-12 and TNF-alpha release in stimulated whole blood cultures was assayed. RESULTS: Whereas the absolute numbers of leukocytes and CD14+ MOs were significantly elevated, HLA-DR expression was suppressed postoperatively. The proportion of TNF-alpha- and IL-12-producing MOs was reduced after surgery. This, however, led to a significant postoperative decrease only in the absolute numbers of peripheral blood IL-12+ MOs. IL-12 secretion was postoperatively reduced in whole blood cultures. The IL-12-synthesizing capacity of IL-12+ MOs was significantly reduced only on d1. CONCLUSIONS: The immediate postoperative period is associated with an increase in the absolute MO numbers and an impairment of MO function, which is reflected in a reduced capacity to synthesize IL-12 and TNF-alpha and a decreased ability to express HLA-DR and present antigens. Whereas the cytokine-producing capacity returns to normal levels on d3, the suppression of HLA-DR expression persists.

Delayed recovery of human leukocyte antigen-DR expression after cardiac surgery with early non-lethal postoperative complications: only an epiphenomenon?

HLA-DR expression on peripheral blood monocytes is reduced after cardiac surgery. Little is known about the reconstitution of HLA-DR expression on peripheral blood monocytes in patients suffering from early non-fatal perioperative complications. We conducted a prospective study to prove whether these complications adversely affect the recovery of HLA-DR expression. Before surgery (d0), on the first (d1), third (d3), fifth (5th) postoperative days, blood samples were collected from 90 patients who underwent elective cardiac surgery with cardiopulmonary bypass (CPB). HLA-DR expression was analysed flow cytometrically. Eleven patients experienced postoperative complications [mechanical ventilation of 24-48 h (n=6); reinstitution of CPB (n=2) intraoperatively; laparotomy (n=1), re-thoracotomy (n=1), re-intubation (n=1) within the first 24 h after surgery]. All patients showed a reduced HLA-DR expression after surgery with nadirs at d1 and d3. Whereas the values increased from d3 to d5 in patients with an uneventful clinical course, HLA-DR expression remained suppressed in patients with complications. HLA-DR expression is reduced after cardiac surgery with CPB. A delayed recovery of HLA-DR expression is seen in patients with early perioperative complications. These non-fatal complications appear to represent a 'second hit' resulting in a prolonged deficiency of the innate immune system. This might predispose to further infectious and septic complications.

In vitro restoration of post-operatively decreased IFN-gamma levels after cardiac surgery and its effect on pro- and anti-inflammatory mediators.

BACKGROUND: A decreased synthesis of interferon gamma (IFN-gamma) by TH 1 lymphocytes after cardiac operations with cardiopulmonary bypass (CPB) is part of the inflammatory response to local operative and systemic traumas. The consequences of this mechanism on the release of pro- and anti-inflammatory cytokines remain unclear. To evaluate the role of IFN-gamma, we added recombinant IFN-gamma to peripheral blood mononuclear cells (PBMCs) on the first post-operative day in an attempt to restore pre-operative values and then measured the release of pro- and anti-inflammatory cytokines in vitro. METHODS: PBMCs of 10 patients scheduled for elective coronary artery bypass grafting (CABG) were obtained pre-operatively (d0) and on the first (d1) and third (d3) post-operative days. The release of IL-6, IL-8, TNF-alpha, IFN-gamma, IL-10, IL-2, and IL-4 was studied after stimulation (48 h) with PHA (phytohemagglutinin) and LPS (lipopolysaccharide) in the absence or presence of recombinant human IFN-gamma. RESULTS: Endogenous IFN-gamma synthesis was suppressed on d1. Adding exogenous IFN-gamma restored IFN-gamma levels to normal on d1 and doubled IFN-gamma levels on d0 and d3. The addition of IFN-gamma increased TNF-alpha levels up to 250% on d1 and IL-2 synthesis by 75% on d1 and d3; the IL-2 levels, however, were still significantly depressed. The addition of recombinant IFN-gamma did not affect the synthesis of IL-6, IL-8, IL-10, and IL-4. CONCLUSIONS: Contrary to our expectations, the in vitro release of IL-6 and IL-8 as well as IL-10 and IL-4 was not influenced by the addition of IFN-gamma. However, TNF-alpha production in isolated PBMC cultures increased significantly on the first post-operative day. This may indicate a hyper-reactivity of PBMCs to IFN-gamma and suggests that the decrease in IFN-gamma synthesis might prevent an excessive stimulation of the non-specific immune system by high TNF-alpha levels after cardiac surgery.

Is interferon gamma suppression after cardiac surgery caused by a decreased interleukin-12 synthesis?

BACKGROUND: The suppression of interferon gamma (IFN-gamma) synthesis after cardiac surgery is discussed as a cause of postoperative immunosuppression that predisposes to postoperative infectious complications. Because several studies have suggested that interleukin-12 (IL-12) production by monocytes and macrophages is reduced after cardiac surgery, this might cause a decrease in IFN-gamma release. To better understand these processes, we assessed the role of IL-12 in IFN-gamma synthesis in vitro before and after cardiac surgery. METHODS: Heparinized whole blood samples were obtained from 20 patients undergoing elective cardiac surgery preoperatively (day 0) and on the first (day 1), third (day 3), and fifth (day 5) postoperative days, and stimulated (24 hours) with staphylococcal enterotoxin B and lipopolysaccharide. Recombinant IL-12 was added at each time point investigated. Interferon-gamma, IL-12, IL-2, IL-4, and IL-5 concentrations and histocompatibility leukocyte antigen-DR (HLA-DR) expression on monocytes and macrophages were assayed by flow cytometry. RESULTS: The HLA-DR expression, IL-12 release, and IFN-gamma synthesis were significantly reduced on day 1, day 3, and day 5. Recovery began on day 3. Interleukin-12 caused a significant increase in IFN-gamma synthesis at each time point. When IL-12 was added, IFN-gamma synthesis returned to preoperative levels on days 3 and 5. CONCLUSIONS: The synthesis of IFN-gamma is significantly reduced after cardiac surgery. The application of IL-12 causes an increase in IFN-gamma synthesis before surgery and a return of IFN-gamma to preoperative levels within a few days after surgery. These findings suggest that postoperative suppression of IFN-gamma release is caused by a decrease in IL-12 synthesis. In addition, IL-12 has a mainly proinflammatory effect both before and after surgery.

Hyporesponsiveness of T cell subsets after cardiac surgery: a product of altered cell function or merely a result of absolute cell count changes in peripheral blood?

OBJECTIVE: The activity of the specific immune system and especially the function of T helper (TH) cells are reduced after cardiac surgery. This decrease is followed by an increase in TH2 cell activity and a delayed recovery of TH1 cell function (TH1/TH2 shift). Neither the underlying cause nor the relationship between the absolute numbers of T lymphocyte subpopulations, the state of activation of these cells and cytokine synthesis in cell culture has been clarified. We conducted a prospective study in order to test the hypothesis that the decrease in specific immunity is not caused by dilution effects but by functional alterations in T cell subsets. METHODS: Blood samples were obtained from 40 patients undergoing elective cardiac surgery with cardiopulmonary bypass (CPB) preoperatively (d0), immediately after surgery (dx), and on the 1st (d1), 3rd (d3) and 5th (d5) postoperative days. The samples were stimulated for 24h with staphylococcal enterotoxin B and lipopolysaccharide. Interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-5 concentrations were measured by flow cytometry using a cytokine bead array kit. We determined white blood cell counts, analysed lymphocyte populations, and assayed human leukocyte antigen (HLA)-DR expression on cluster of differentiation (CD)4+ and CD8+ lymphocytes. Cytokine concentrations were corrected to preoperative absolute numbers of T helper cells. RESULTS: Leukocyte counts were elevated during the entire postoperative course with a maximum on dx. Absolute lymphocyte counts and especially the T cell subpopulations significantly increased immediately after surgery, then decreased to a minimum on d1 and increased again until they returned to preoperative levels on d3. The release of IFN-gamma, IL-2 and IL-4 was significantly reduced from dx to d5 with a minimum on d1. IL-5 was significantly reduced on dx and d1. When the concentrations were corrected to preoperative TH lymphocyte levels, IL-2 and IL-5 synthesis was significantly reduced only on dx and IL-4 release only on dx and d1. By contrast, IFN-gamma synthesis decreased postoperatively and remained suppressed until d5 with a minimum on d1. Only on d1 did an increase in HLA-DR expression give evidence of a change in the state of TH cell activation. CONCLUSIONS: The number of immune cells of the specific and the non-specific immune system is not reduced in the immediate postoperative period. Haemodilution thus has no detectable effect on immune function at this time point. Beginning on d1, the function of specific immune cells, especially TH lymphocytes, is severely suppressed. This functional alteration appears not to be preceded by T cell activation during CPB. Although TH cell activity begins to increase on d1, cytokine synthesis is reduced. When cytokine synthesis is corrected to the absolute number of TH cells in culture, there is strong evidence for an increase in TH2 cell activity. On the whole, these results corroborate the hypothesis of a TH1/TH2 shift that is primarily caused by an alteration of TH1 function. Neither haemodilution nor a preceding activation plays a major role.

Pro-inflammatory cytokines after different kinds of cardio-thoracic surgical procedures: is what we see what we know?

OBJECTIVE: Due to the combination of local trauma, extracorporeal circulation (ECC), and pulmonary and myocardial reperfusion, cardiac surgery leads to substantial changes in the immune system and possibly to post-operative complications. Procedures without ECC, however, have failed to demonstrate clear advantages. We hypothesized that ECC is far less important in this context than the reperfusion/reventilation of the lung parenchyma and the surgical trauma. We therefore conducted a prospective observational study to compare immune reactions after cardiac operations with those after thoracic surgery. METHODS: Serum levels of pro-inflammatory interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha as well as C-reactive protein (CRP), lipoprotein-binding protein (LBP) and procalcitonin (PCT) were measured pre-operatively (d0), at the end of the operation (dx), 6h after the operation (dx+), on the 1st (d1), 3rd (d3), and 5th (d5) post-operative days in 108 patients (pts) undergoing elective coronary artery bypass grafting (CAB) with ECC (n=42, CPB CAB), off-pump coronary artery bypass surgery (n=24, OP CAB) without ECC or thoracic surgery (n=42, TS). RESULTS: After cardiac surgery (CS), IL-6 and IL-8 increased and reached a maximum on dx+. IL-6 returned to baseline values at d3, whereas IL-8 remained elevated until d5. No difference was found between OP CAB and CPB CAB patients. In the TS patients, IL-6 increased later (dx+) and absolute levels were lower than in the CS patients. No increase in IL-8 was noted in the TS patients. Due to the high variation in the results obtained in all three groups, there was no significant change in TNF-alpha. A comparison of TS, OP CAB, and CPB CAB revealed that the CS patients had higher levels on d0, dx, d3, and d5. Serum levels of CRP, LBP, and IL-2R increased from dx+ to d5 in all groups and reached maximum values on d3. Whereas we found no difference in CRP and IL-2R between the groups, LBP levels were significantly higher from dx+ to d3 after OP CAB. PCT was elevated from dx+ to d3 in all pts. Similar levels were noted for the TS and OP CAB patients. The CPB CAB patients showed the highest levels. CONCLUSIONS: Surgical trauma and reperfusion injury appear to represent the predominant factors resulting in immunologic changes after cardiac surgery. Cardiopulmonary bypass (CPB) may be less important for immune response and acute-phase reactions than previously suspected. In addition, our data indicate a relationship between IL-6 synthesis and the degree of surgical trauma. IL-8 appears to be elevated only after cardiac surgery whereas PCT liberation depended on the use of ECC.

Proinflammatory and antiinflammatory cytokines after cardiac operation: different cellular sources at different times.

BACKGROUND: Cardiac operation produces substantial alterations within the immune system, which possibly predispose postoperative complications. However, the interplay between proinflammatory and antiinflammatory reactions and the cells involved in this process are not completely clear. Therefore, we investigated serum levels, as well as synthesis patterns, of proinflammatory and antiinflammatory cytokines. METHODS: Serum levels and production of interleukin (IL) IL-5, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma, using a mixed cell culture, (ie, monocytes, macrophages, and lymphocytes), as well as a purified lymphocyte culture were measured preoperatively (day 0), on postoperative day 1, on postoperative day 3, and on postoperative day 5 in 25 patients undergoing cardiac operations and were compared with 10 healthy volunteers. RESULTS: Serum level and mixed cell culture, production of IL-6, tumor necrosis factor-alpha, and IL-10 increased on postoperative day 1, but decreased in lymphocyte culture. Base line values were reached on postoperative day 5. Interferon-gamma serum levels remained unchanged, whereas IL-5 serum levels increased on postoperative days 3 and 5. Cell culture synthesis showed a significant suppression for both mediators in both cell cultures, which returned to baseline on postoperative day 3 in mixed cell culture. Interferon-gamma production by lymphocytes was suppressed until postoperative day 5, whereas IL-5 returned to preoperative values on postoperative day 5. CONCLUSIONS: Cardiac operation induces a biphasic immune response. The first phase (postoperative day 1) appears to represent the proinflammatory and antiinflammatory reaction of the innate immune system returning to base line on postoperative day 3. The second phase (postoperative day 5) may represent the response of the adaptive immune system and is characterized by an antiinflammatory type of reaction. This may explain why the systemic inflammatory response occurs immediately after cardiac operation, whereas infections occur later.