Dhanonjoy C. Saha

Monocyte response to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis

Exposure to endotoxin produces a state of macrophage hyporesponsiveness on subsequent stimulation. Monocytes in patients with septic shock demonstrate a similar hyporesponsiveness to endotoxin. The purpose of this study was to examine whether this state of hyporesponsiveness extends to other inflammatory stimuli and the relationship of this state to cell surface receptor expression and the release of anti-inflammatory cytokines. Twelve normal volunteers, 10 patients with severe sepsis, and 9 patients with septic shock were included in the study. Monocytes from each subject were isolated and stimulated with lipopolysaccharide (LPS), staphylococcal enterotoxin B (SEB), and phorbol myristate acetate (PMA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). Serum levels of transforming growth factor-beta1 (TGF-beta1), prostaglandin E2 (PGE2), and interleukin-10 (IL-10) were also measured by ELISA. The expression of monocyte CD14 and HLA-DR in whole blood were measured by flow cytometry. Patients with septic shock demonstrated significantly decreased TNF-alpha and IL-1beta release as compared with normal subjects in response to LPS. In response to SEB, patients with sepsis and patient with septic shock demonstrated significantly decreased release of TNF-alpha and IL-1beta. Significant decreases in TNF-alpha release were found in the patients with septic shock after PMA stimulation. There were no significant differences in the monocyte response to the different stimuli between patients with gram-positive sepsis and gram-negative sepsis. HLA-DR expression was significantly decreased in patients with septic shock (58 +/- 9 fluorescence units (flU)) as compared with normal subjects (102 +/- 14 flU) (p < 0.05). No differences in CD14 expression were observed. IL-10 levels were significantly increased in patients with sepsis (16 +/- 4 pg/ml) and in patients with septic shock (42 +/- 15 pg/ml) and were detectable in 1 normal subject. TGF-beta1 levels were decreased in patients with septic shock (25 +/- 6 pg/ml) as compared with those in normal subjects (37 +/- 2 pg/ml)(p < 0.05). PGE2 levels were significantly increased in patients with septic shock and patients with sepsis. These data are consistent with a more generalized monocyte hyporesponsiveness to bacterial toxins that may be related to altered cell surface receptor expression and the release of anti-inflammatory cytokines.

Role of interleukin-10 in monocyte hyporesponsiveness associated with septic shock

ObjectivesThe purpose of this study was to examine the pattern of tumor necrosis factor (TNF)-&agr; and interleukin (IL)-10 release in endotoxin-stimulated septic monocytes and to determine the role of IL-10 and transforming growth factor (TGF)-&bgr; in monocyte hyporesponsiveness during septic shock. DesignMonocytes isolated from ten healthy controls and ten patients with septic shock were incubated with endotoxin and cytokine release was assessed. Next, normal monocytes were incubated with either normal or septic serum and stimulated with endotoxin. Finally, normal monocytes were incubated with septic serum either with anti-IL-10 antibodies or anti-TGF-&bgr; antibodies and then stimulated with endotoxin. MeasurementsTNF-&agr;, IL-10, and TGF-&bgr; levels were measured in the serum and in culture supernatants by enzyme-linked immunosorbent assay. SettingResearch laboratory. Main ResultsIL-10 and TNF-&agr; levels were significantly increased in septic serum, whereas TGF-&bgr; levels were not different from controls. Normal monocytes increased TNF-&agr; and IL-10 release in response to endotoxin. In contrast, septic monocyte TNF-&agr; release was attenuated in response to endotoxin (1.8 ± 0.5 vs. 1.0 ± 0.4 ng/mL, stimulated vs. baseline), whereas IL-10 release increased significantly from baseline (173 ± 91 vs. 8 ± 4 pg/mL, stimulated vs. baseline). Incubation of normal monocytes with septic serum attenuated TNF-&agr; release in response to endotoxin (32% ± 8% of normal serum;p < .01), whereas IL-10 release was increased (285% ± 84% of normal serum;p < .05). When normal monocytes were incubated with septic serum combined with anti-IL-10 antibodies, TNF-&agr; release increased significantly to 75% ± 17% of normal serum (p < .05 vs. septic serum alone). Incubation of normal monocytes with anti-TGF-&bgr; antibodies did not significantly affect either TNF-&agr; or IL-10 release in response to endotoxin. ConclusionMonocytes from patients with septic shock exhibit persistent IL-10 release at a time when TNF-&agr; release is down-regulated. The continued release of IL-10 may contribute to impairment of monocyte proinflammatory cytokine release and the development of immune dysfunction in septic shock.

Microvascular response in patients with cardiogenic shock.

Objective: To examine the mechanisms contributing to decreased microvascular blood flow in cardiogenic shock by comparing patients with cardiogenic shock with critically ill controls and with patients with septic shock. Design: Prospective, consecutive entry of patients meeting the criteria for septic shock, cardiogenic shock, and critical illness without coexisting infection or shock. Setting: University hospital, medical intensive care unit, coronary care unit, and respiratory care unit. Patients: Eight patients with cardiogenic shock secondary to acute myocardial infarction, six critically ill controls, and six patients with septic shock. Measurements and Main Results: Forearm blood flow was measured at rest and during reactive hyperemia by venous air plethysmography. Red cell deformability was determined by filtration. Leukocyte aggregation was detected by the leukergy test. Neutrophil CD11b/CD18 expression and soluble intercellular adhesion molecule‐1 levels were also measured. In cardiogenic shock, forearm arterial resistance was significantly increased at rest and during reactive hyperemia compared with controls and patients with septic shock. The response to reactive hyperemia was attenuated in cardiogenic and septic shock patients, as measured by the absolute change in forearm blood flow from baseline, which was significantly less as compared with controls (p < .01). The percent change in forearm blood flow during reactive hyperemia compared with forearm blood flow at rest was significantly lower in cardiogenic shock (60 ± 10) and in septic shock (50 ± 11) compared with controls at baseline (145 ± 20; p < .01). Red cell deformability was significantly decreased in cardiogenic shock (1.2 ± 0.2 mL/min; p < .05) and septic shock (1.1 ± 0.2 mL/min; p < .05), compared with controls (1.8 ± 0.1 mL/min). Neutrophil CD11b/CD18 expression, leukergy, and serum intercellular adhesion molecule‐1 levels in cardiogenic shock patients were not significantly different from controls. Conclusion: These data suggest that the response to reactive hyperemia is attenuated in cardiogenic shock. This appears to reflect increased vasoconstriction and an impaired capacity for vasodilation. Decreased erythrocyte deformability may also be important in limiting systemic microvascular flow. However, evidence supporting a role for neutrophil‐endothelial cell interactions was not observed.

Relationships between plasma cytokine concentrations and leukocyte functional antigen expression in patients with sepsis.

Objective: To determine the relationships between cytokine concentrations and alterations in leukocyte functional antigen expression in sepsis. Design: Prospective, cross‐sectional study. Setting: Respiratory, coronary, and medical intensive care units in a university hospital. Patients: Forty subjects consisting of: a) patients with severe sepsis, b) patients with sepsis, c) critically ill nonseptic patients, and d) normal controls. Interventions: None. Measurements: Plasma concentrations of interleukin (IL)‐1&bgr;, IL‐6, IL‐8, IL‐10, interleukin‐1 receptor antagonist (IL‐1Ra), and tumor necrosis factor (TNF)‐&agr; were determined by enzymelinked immunosorbent assay (ELISA). Peripheral blood monocyte HLA‐DR and CD14 expression and neutrophil CD11b expression were determined by flow cytometry. Measurements were taken within 24 hrs of admission to the intensive care unit and/or clinical presentation. Main Results: Significantly increased plasma IL‐6, IL‐8, IL‐10, and TNF‐&agr; concentrations were observed in the severe sepsis group compared to normal controls. Increases in IL‐1Ra were not significant. Monocyte HLA‐DR expression, significantly decreased in patients with severe sepsis, was correlated both with IL‐6 ( p < .005) and IL‐8 concentrations ( p < .001). Both of these cytokines had close correlations to Acute Physiology and Chronic Health Evaluation (APACHE) II scores which were also correlated with monocyte HLA‐DR. Neutrophil CD11b, which was increased in all infected patients, was significantly correlated with the ratio between IL‐1 and IL‐1Ra concentrations ( p < .001). The percent of CD14+ monocytes was lowest in patients with severe sepsis and showed a significant covariate effect from IL‐8 concentrations ( p < .001). Conclusion: These findings suggest that the expression of specific functional molecules on peripheral blood leukocytes is variably related to the net production of certain monokines in sepsis. (Crit Care Med 1994; 22:1595–1602)

Mechanisms of platelet-neutrophil interactions and effects on cell filtration in septic shock

We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock. Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers. Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock. Cell adhesion was assessed by filtration through 5-&mgr;m pore filters and by flow cytometry. Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIIa, P-selectin, ICAM-2, CD11a, CD11b, and CD18. The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 ± 1.0 mmHg vs. 14 ± 1.0 mmHg;P < 0.05). The difference between the Pi of cells suspended in SPPP or NPPP (&Dgr;Pi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the &Dgr;Pi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01). The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies. The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 ± 130 lfu vs. 295 ± 35 lfu;P < 0.05). The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil. We conclude that platelet-neutrophil aggregation is increased in septic shock. This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors. The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions.

Monophosphoryl lipid A protects against gram-positive sepsis and tumor necrosis factor.

Monophosphoryl lipid A (MPL) is a less toxic derivative of lipid A that enhances survival from endotoxemia. This study examined whether MPL induced resistance to Gram-positive sepsis and cytokines. Mice were administered MPL or saline (phosphate-buffered saline) and challenged 24 h later with live Staphylococcus aureus (SA), staphylococcus enterotoxin B (SEB), toxic shock syndrome toxin (TSST-1), and tumor necrosis factor (TNF). Survival was determined at 72 h. A separate set of animals was phlebotomized for determination of cytokines. MPL increased survival from S. aureus bacteremia from 20 to 87% (p < .05). Interleukin-6 (IL-6) and interleukin-1 (IL-1) and TNF were also significantly decreased. SEB and TSST survival were enhanced from 10 to 90% (p < .05). In SEB-treated animals, TNF and IL-6 levels were significantly decreased. Survival from TNF infusion was increased from 20 to 100% with MPL, however, no significant differences in cytokines were observed. These data suggest that MPL induces resistance to Gram-positive sepsis and cytokine-mediated activity.

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen‐presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less‐toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O2−) and hydrogen peroxide (H2O2), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O2− and H2O2 was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 μg/ml MPL, the same concentration that induced the maximum release of H2O2 in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IκBα, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.

Monophosphoryl lipid A stimulated up-regulation of nitric oxide synthase and nitric oxide release by human monocytes in vitro

Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) with reduced toxicity which has been shown to modulate various immune functions in monocytes. We examined whether human monocytes can be stimulated to produce nitric oxide (NO) and its catalytic enzyme nitric oxide synthase (NOS). Monocytes were stimulated with LPS or MPL and both NOS and NO (as nitrite) production were measured. MPL at high doses (> 100 micrograms/ml) stimulated monocytes to release NO that was significantly greater than both the control and LPS-treated monocytes (p < 0.05). NO release by control cells and the LPS treated cells was not significantly different. Both arginase and N-monomethyl arginine (NMLA) inhibited the MPL stimulated release of NO (p < 0.01). MPL significantly increased inducible NOS (iNOS) expression as measured by both fluorescent microscopy and flow cytometry (p < 0.05). Similarly, both soluble NOS (sNOS) and particulate NOS (pNOS) activity were significantly up-regulated by MPL (p < 0.05). Significant correlations were found between pNOS expression and sNOS release (r = 0.72, p < 0.0001) and between 12 h NO release and sNOS production (r = 0.44, p < 0.005). These experiments confirm that human monocytes can be stimulated with MPL to produce NO in vitro and suggest that up-regulation of pNOS does not preclude NO release.

Diphosphoryl lipid A from Rhodopseudomonas sphaeroides induces tolerance to endotoxic shock in the rat.

ObjectivesTo examine the hemodynamic effects of diphosphoryl lipid A from Rhodopseudomonas sphaeroides and to examine the ability of this substance to induce tolerance to endotoxic shock. DesignRandomized, prospective, controlled study comparing the hemodynamic actions of R. sphaeroides diphosphoryl lipid A to those effects of lipopolysaccharide from Salmonella minnesota, followed by a prospective, randomized, controlled study comparing pretreatment with R. sphaeroides diphosphoryl lipid A and phosphate-buffered saline in the induction of tolerance to endotoxic shock. SettingLaboratory of the Section of Critical Care Medicine at a University Hospital. SubjectsMale Sprague-Dawley rats. InterventionsEight rats were randomized to receive intravenous R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight or S. minnesota lipopolysaccharide, 0.5 mg/100 g body weight. Ten rats were then randomized to receive R. sphaeroides diphosphoryl lipid A, 0.5 mg/100 g body weight, or phosphate-buffered saline intravenously 48 hrs before receiving S. minnesota lipopolysaccharide, 5 mg/100 g body weight, by intravenous infusion. Measurements and Main ResultsCardiac index was significantly decreased from baseline in rate treated with lipopolysaccharide; there was no significant change in the R. sphaeroides diphosphoryl lipid A group. Peak circulating tumor necrosis factor (TNF) concentrations in the lipopolysaccharide-treated rats were higher than in R. sphaeroides diphosphoryl lipid A-treated rats (3.1 ± 1.0 vs. 1.5 ± 0.4 ng/mL). R. sphaeroidesdiphosphoryl lipid A significantly attenuated lipopolysaccharide-induced changes in mean arterial pressure and cardiac index. At baseline, there was no significant difference in serum TNF concentrations between rats pretreated with R. sphaeroides diphosphoryl lipid A and those rats pretreated with phosphate-buffered saline. TNF levels peaked at 1 hr post-lipopolysaccharide infusion at 4.3 ± 0.6 ng/ mL in the phosphate-buffered saline group and at 2.0 ± 0.5 ng/mL in the R. sphaeroides diphosphoryl lipid A group (p < .02). Four of five rats pretreated with R. sphaeroides diphosphoryl lipid A survived endotoxic shock, whereas none of the phosphate-buffered saline-pretreated rats survived (p = .05). ConclusionsThese observations are consistent with previous reports of the limited toxic effects of R. sphaeroides diphosphoryl lipid A and suggest that this molecule retains the ability to induce tolerance to endotoxic shock. (Crit Care Med 1993; 21:753–758)

Endogenous free radical generating sources are involved in smoking-mediated dysfunction of nitric oxide biosynthesis in human coronary artery endothelial cells: An in vitro demonstration

Background: Vulnerable plaques contain abundant macrophages. We developed a novel photodynamic agent, chlorin, conjugated with maleylated albumin, (ce6-mal-alb) that concentrates in macrophage-rich plaques and has a high fluorescent yield. As such, we tested hypothesis that experimental atherosclerotic lesions (ATHERO) can be detected using ce6-mal-alb and an intravascular fluorescence spectroscopy catheter. Methods: ATHERO were induced in New Zealand rabbits by infradiaphragmatic aortic balloon-injury followed by high cholesterol diet. At IO weeks, ce6-mal-alb was administered to 7 atherosclerotic and 7 control animals. 24 hours later, aortic uptake of the ce6mal-alb uptake was measured in situ, using an intravascular fluorescence spectroscopy catheter. Thereafter, the aortas were excised and dissolved in NaOH/SDS for spectrofluorimetric determination of ce6 content. Results: Intravascular measurements of ce6-mal-alb fluorescence were higher in ATHERO vs. non-athero segments, (8.8i4.8 vs. 2.2k2.2 X103AU, P<O.OOl, figure). Further, ce6-mal-alb concentration was higher within the ATHERO aortas (5.2t3.2 vs. 1.9+1.2, ce6 fluorescencelgm tissue x IOs, p=<O.Ol). Conclusion: CeG-mal-alb can be employed for intravascular characterization of atherosclerotic plaques. Because this novel PDT compound is selectively toxic to macrophages when light-activated, this agent may be useful for both the detection and therapeutic modification of macrophage-rich plaques.