Zoltán Spolarics

Superoxide generation by Kupffer cells and priming of neutrophils during reperfusion after hepatic ischemia.

The objective of this study was to identify the cellular source of the vascular oxidant stress in hepatic ischemia-reperfusion injury in male Fischer rats. Nonparenchymal cells (Kupffer cells, endothelial cells) and neutrophils were isolated from postischemic liver lobes by collagenase-pronase digestion followed by centrifugal elutriation. The spontaneous and stimulated generation of superoxide by these cells were subsequently quantified in vitro. Large Kupffer cells from the postischemic lobes spontaneously generated 300% more superoxide than similar cells from control animals. No difference in spontaneous superoxide formation was found when the small Kupffer cells were compared. No other cells isolated from the postischemic lobes or control liver including neutrophils released any detectable superoxide spontaneously. In contrast, small Kupffer cells and neutrophils from the postischemic liver generated significantly more superoxide after stimulation with phorbol ester or opsonized zymosan than the controls. The considerably higher response with zymosan stimulation compared to phorbol ester indicates a particular priming for a receptor-mediated signal transduction pathway during reperfusion. These studies demonstrate that Kupffer cells are the principal source of the oxidant stress during the initial reperfusion phase after hepatic ischemia. The priming of neutrophils during this time may be an important factor for the later neutrophil-induced injury phase.

Superoxide generation by neutrophils and Kupffer cells during in vivo reperfusion after hepatic ischemia in rats.

Kupffer cells and polymorphonuclear leukocytes (PMNs) contribute to the severe reperfusion injury of the liver after ischemia at different time points. The objective of this study was to identify die cellular source(s) of reactive oxygen formation during the PMN‐ induced injury phase. Kupffer cells and PMNs were isolated from the liver after 45 min of ischemia and 5 h or 24 h of reperfusion using collagenase‐pronase digestion and a centrifugal elutriation method. Spontaneous superoxide anion (O2−) formation by large Kupffer cells (basal value 0.65 ± 0.16 nmol/h/106 cells) was increased (up to 550%) during the entire reperfusion period. No enhanced O2− generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of O2− spontaneously (0.25 ± 0.05 nmol O2−/h/106 cells), but hepatic PMNs generated significantly more superoxide: 1.90 ± 0.58 nmol O2−/h/106 cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced O2− formation during reperfusion; the priming effect was consistantly higher for stimulation with opsonized zymosan (receptor‐ mediated signal transduction pathway) compared to phor‐ bol myristate acetate (protein kinase C activation). Our data support the hypothesis that PMNs and large Kupffer cells are predominantly responsible for the postischemic oxidant stress during the later reperfusion injury phase after hepatic ischemia in viva.

ENDOTOXEMIA, PENTOSE CYCLE, AND THE OXIDANT/ANTIOXIDANT BALANCE IN THE HEPATIC SINUSOID

During the innate immune response, excessive release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver and other tissues. The consequence of oxidative stress is determined by the status and adaptive changes of antioxidant pathways. In this review, we present evidence that the synchronized response of hepatic sinusoidal endothelial cells, the primary sites of phagocyte attachment, plays an important role in defense against phagocyte‐derived ROS. An essential component of the metabolic adaptation of hepatic sinusoidal cells to lipopolysaccharide (LPS)‐induced oxidative stress is the stimulated expression of glucose‐6‐phosphate dehydrogenase (G6PD), the key enzyme of the pentose cycle (hexose monophosphate shunt, HMS). All major ROS‐metabolic enzymes, i.e., glutathione peroxidase, glutathione reductase, catalase, superoxide dismutases, NADPH oxidase, and nitric oxide synthase, directly or indirectly depend on NADPH, which is produced in the HMS in these cells. The functional significance of up‐regulated HMS within a particular cell type depends on the accompanying adaptive changes in ROS‐metabolizing enzymes. In LPS‐activated Kupffer cells, the elevated expression of glucose transporter GLUT1 and G6PD mainly serves primed production of superoxide anion, hydrogen peroxide, and nitric oxide. In sinusoidal endothelial cells, the LPS‐induced response pattern of glucose‐ and ROS‐metabolizing enzymes results in elevated ROS detoxifying capacity. The described studies also suggest the existence of an intercellular oxidant balance between prooxidant Kupffer cells and antioxidant endothelial cells in the hepatic micro‐environment. Maintenance of the intercellular oxidant/antioxidant balance between phagocytes and endothelial cells may represent an important mechanism protecting the hepatic parenchyma against exogenous oxidative stress during the inflammatory response. J. Leukoc. Biol. 63: 534–541; 1998.

Depressed interleukin-12-producing activity by monocytes correlates with adverse clinical course and a shift toward Th2-type lymphocyte pattern in severely injured male trauma patients

OBJECTIVE To determine the effect of major trauma on the cytokine-producing activity of monocytes and CD4+ T cells in a homogeneous cohort of patients as well as to determine the relationship between monocyte and T-lymphocyte responses and clinical outcome. SETTINGS Surgical intensive care units of a trauma center and flow cytometry and experimental laboratories at a teaching hospital. DESIGN Prospective cohort clinical study with measurements of white cell cytokine-producing activity on days 2, 5, and 10 postinjury. The number of cytokine-producing CD14+ monocytes, CD4+, and CD8+ T cells were determined in whole blood using flow cytometry combined with the intracellular cytokine staining method. Basal and lipopolysaccharide-stimulated interleukin (IL)-12, tumor necrosis factor-alpha, IL-6, and IL-1alpha production by monocytes as well as basal and phorbol 12-myristate 13-acetate plus ionomycin-stimulated interferon-gamma, IL-4, and tumor necrosis factor-alpha production by T cells were determined on days 2, 5, and 10 postinjury and compared with similar measurements made in healthy control subjects. PATIENTS Twelve randomly selected black, male patients were enrolled in the study: mean injury severity score, 26; mean age, 35 yrs; mean Glasgow Coma Scale score, 13; systemic inflammatory response syndrome, 92%; sepsis, 42%; bronchial infection, 42%; and adult respiratory distress syndrome 25%. MAIN RESULTS After lipopolysaccharide stimulation, the number of IL-12-, tumor necrosis factor-alpha-, IL-1alpha-, and IL-6-producing CD14+ monocytes was 40% to 70% lower in trauma patients on postinjury days 2, 5, and 10 than in healthy control subjects. After phorbol 12-myristate 13-acetate stimulation, the number of IL-4-producing CD4+ cells increased three-fold in the trauma patients compared with healthy control subjects. In contrast, the number of interferon-gamma- or tumor necrosis factor-alpha-producing CD4+ and CD8+ T cells was not different between the patients and control subjects. The Th1/Th2 ratio was significantly lower in patients on all postinjury days than in the control subjects. A statistically significant inverse correlation was found between the number of IL-12-producing monocytes and IL-4-producing CD4+ T cells in trauma patients (p =.007, r2 =.47). This correlation was absent in control subjects. The degree of depressed capacity of monocyte IL-12 production on day 2 postinjury showed a statistically significant correlation with the development of adult respiratory distress syndrome, sepsis, or infections and also with the duration of systemic inflammatory response syndrome and sepsis. CONCLUSIONS Major trauma results in an early and marked decrease in monocyte cytokine-producing activity. The trauma-induced depression in IL-12 production by the mononuclear phagocyte system may promote T-cell commitment toward a Th2 pattern early after trauma. The appearance of the Th2 pattern is the result of elevated numbers of IL-4-producing cells without major alterations in T-cell interferon-gamma-producing capacity. The degree of alterations in monocyte and T-cell responses on day 2 postinjury correlates with the development of adverse clinical outcomes and the subsequent duration of the inflammatory response.

Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function

Adenosine is an immunosuppressive nucleoside, and adenosine A2A receptors inhibit T‐cell activation. We investigated the role of A2A receptors in regulating T helper (Th)1‐ and Th2‐cell development and effector function. A2A‐receptor stimulation suppressed the development of T‐cell receptor (TCR) ‐stimulated naive T cells into both Th1 and Th2 cells, as indicated by decreased IFN‐γ production by cells developed under Th1‐skewing conditions and decreased interleukin (IL) −4, IL5, and IL10 production by cells developed under Th2‐skewing conditions. Using A2A receptor‐deficient mice, we demonstrate that A2A receptor activation inhibits Th1‐ and Th2‐cell development by decreasing the proliferation and IL2 production of naive T cells, irrespective of whether the cells are expanded under Th1‐ or Th2‐skewing environment. Using in vivo established Th1 and Th2 cells, we further demonstrate the nonselective nature of A2A receptor‐mediated immunosuppressive effects, because A2A receptor activation decreased IFN‐γ and IL4 secretion and mRNA level of TCR‐stimulated effector Th1 and Th2 cells, respectively. A2A receptor mRNA expression in both Th1 and Th2 effector cells increased following TCR stimulation. In summary, these data demonstrate that A2A receptor activation has strong inhibitory actions during early developmental, as well as late effector, stages of Th1‐ and Th2‐cell responses.—Csóka, B., Himer, L., Selmeczy, Z., Vizi, E. S., Pacher, P., Ledent, C., Deitch, E. A., Spolarics, Z., Németh, Z. H., Haskó, G. Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function. FASEB J. 22, 3491–3499 (2008)

A2B Adenosine Receptors Protect against Sepsis-Induced Mortality by Dampening Excessive Inflammation

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein–coupled receptors and can regulate the host’s response to sepsis. In this study, we studied the role of A2B adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A2B knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-κB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A2B receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A2B receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A2B receptor-deficient mice. These results indicate that A2B receptor activation may offer a new therapeutic approach for the management of sepsis.

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host’s response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5′-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

Effect of trauma-hemorrhagic shock on red blood cell deformability and shape.

Previous work in our laboratory has demonstrated a decrease in red blood cell (RBC) deformability in sepsis. This has not been studied following hemorrhagic shock. We tested the hypotheses that hemorrhagic shock, associated with soft tissue trauma, leads to decreased RBC deformability and that this is related to alterations in the resting shape of the RBC. Elongation index (EI), a measure of RBC deformability, was determined over a range of shear stresses from 0.3 to 30 Pa in 26 male rats before and at various times after 90 min of hemorrhagic shock. RBC resting shape was determined by scanning electron microscopy. The data demonstrate that EI decreased significantly at the end of shock (before resuscitation), and remained below normal throughout the 6-h postshock period. Eight of the 26 animals decompensated during shock, requiring return of a portion of the shed blood to maintain a mean arterial pressure of 30–40 mmHg. Four of eight decompensated animals died before the end of the study period, compared with none of the compensated rats. The decompensated rats had significantly lower EI at 0.3 Pa by the end of the shock period (0.050 ± 0.009) than the compensated shock group (0.058 ± 0.006;P < 0.05). RBC shape alterations were first demonstrated at the end of the shock period and persisted throughout the 6-h postshock resuscitation period. These data indicate that trauma and hemorrhagic shock cause RBC shape alterations and a significant decrease in RBC deformability, which becomes manifested during the shock period and persists for at least 6 h postshock. Additionally, a direct relationship appears to exist between the magnitude of the physiologic insult and the degree of RBC damage.

Increased incidence of sepsis and altered monocyte functions in severely injured type A- glucose-6-phosphate dehydrogenase-deficient African American trauma patients.

ObjectiveTo determine whether trauma patients with the common, type A− glucose-6-phosphate dehydrogenase (G6PD) deficiency have an aggravated inflammatory response, increased incidence of septic complications, and/or more profound alterations in leukocyte functions compared with nondeficient trauma patients. SettingsIntensive and surgical care units of a trauma center and flow cytometry and experimental laboratories at a teaching university hospital. DesignProspective cohort clinical study with measurements on days 2 and 5 postinjury. Monocyte and neutrophil oxidant content, apoptosis, and CD11b expression and plasma cytokine levels were compared between G6PD-deficient and nondeficient patients. PatientsA total of 467 male African American trauma patients were screened for the deficiency. Forty-four type A−202/376 G6PD-deficient patients were identified and enrolled in the study; 43 nondeficient patients were also enrolled and were matched by age, clinical criteria of injury severity, and type of trauma. Main ResultsAfter severe injury (Injury Severity Score, ≥16), 50% of the deficient and 6.2% of nondeficient patients developed sepsis with positive bacterial blood cultures. In deficient patients, the frequency of bronchial (75%) and wound infections (25%) was also increased compared with nondeficient patients (32% and 0%). The durations of systemic inflammatory response syndrome, Sepsis Syndrome, and days on antibiotics were three times longer in deficient than in nondeficient individuals. However, adult respiratory distress syndrome occurred in 37% of both groups. Anemia was more severe in the deficient than nondeficient patients from day 10 posttrauma. On day 5, the peroxide content was doubled, apoptosis was decreased, and CD11b expression was increased in monocytes from deficient patients compared with cells from nondeficient patients. On day 5, the plasma interleukin (IL)-10 concentration was significantly lower in deficient than nondeficient patients, whereas tumor necrosis factor-&agr;, IL-6, and IL-8 levels were similar. After moderate injuries (Injury Severity Score, 9–16), the deficiency was not associated with adverse clinical effects, and the trauma-induced changes in leukocyte function were similar in deficient and nondeficient patients. ConclusionsThe common type A− G6PD deficiency predisposes septic complications and anemia in trauma patients after severe injuries as defined by an Injury Severity Score of ≥16. This adverse clinical course is accompanied by altered monocyte functions manifested as augmented oxidative stress, a decreased apoptotic response, increased cell adhesion properties, and a diminished IL-10 response.

CB2 Cannabinoid Receptors Contribute to Bacterial Invasion and Mortality in Polymicrobial Sepsis

Background Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB2 receptors on immune cells. Here we examined the role of CB2 receptors in regulating the hosts response to sepsis. Methods and Findings The role of CB2 receptors was studied by subjecting CB2 receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB2 receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB2 receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF)-κB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB2 receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b+ and CD19+ cells during CLP. Conclusions Taken together, our results establish for the first time that CB2 receptors are important contributors to septic immune dysfunction and mortality, indicating that CB2 receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.