Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Mechanisms for cell activation and its consequences for biorheology and microcirculation: Multi-organ failure in shock.

Activation of cells in the vascular compartment causes profound alteration of cell rheological properties with impairment of the microcirculation and initiation of inflammatory reactions. Many cardiovascular diseases have been shown to be associated with cell activation and inflammation. While this situation offers the opportunity for new interventions against the deleterious effects of cell activation, there is the need for a better understanding of the mechanisms that lead to cell activation in the first place. We review here several mechanisms for cell activation in the circulation. We show that in shock, a condition associated with severe forms of cell activation, humoral cell activation factors can be detected in plasma. Further analysis indicates that the source of these humoral activators may be due to the action of pancreatic digestive enzymes in the intestine. Ischemia may serve to open the intestinal brush border and permit entry of pancreatic enzymes into the wall of the intestine to initiate self digestion. In this process low molecular weight but potent cell activators are produced which may escape via the intestinal circulation and the lymphatics into the general circulation. Inhibition of pancreatic enzymes in the lumen of the intestine leads to complete attenuation of humoral activator production as well as many of the deleterious sequelae that accompany shock, such as inflammation and multi-organ failure. We outline a method to carry out biochemical isolation of the cell activators derived from pancreatic enzymes. This analysis shows that there are multiple species of cell activators above and beyond currently known species, many of which have molecular weights below 3000 Da. Identification of the mechanisms that lead to cell activation is an important part to understand the mechanisms that lead to alterations of rheological properties of blood cells in disease and dysfunction of the endothelium and parenchymal cells. Our current evidence suggests that pancreatic digestive enzymes and tissue enzymes may play a central role in humoral activator production.

Pancreatic enzymes and microvascular cell activation in multiorgan failure.

Cell activation in the microcirculation leads to an inflammatory cascade and is accompanied by many cardiovascular complications. There is a need to identify the trigger mechanisms that lead to the production of in vivo activating factors. We review here mechanisms for cell activation in the microcirculation and specifically the production of humoral cell activators in physiological shock. The elevated levels of activating factors in plasma could be traced to the action of pancreatic enzymes in the ischemic intestine. New interventions against the production of the activators are proposed. The evidence suggests that pancreatic enzymes in the ischemic intestine may attack several tissue components and generate cellular activators that are associated with multiorgan dysfunction in physiological shock.

Intratracheal administration of anaphylatoxin C5a potentiates antigen-induced pulmonary reactions through the prolonged production of cysteinyl-leukotrienes.

The effects of intratracheal administration of anaphylatoxin C5a on airway inflammation have been studied using two sources of material, zymosan activated serum (ZAS) and purified rat C5a des Arg, in order to determine the influence of complement activation on allergic airway disorders.The intratracheal administration of ovalbumin (OA) to OA-sensitized rats generated two phases of airway response, an immediate airway response (IAR) occurring within 15 min and a late airway response (LAR) beginning 4-6 h after the allergen challenge. The simultaneous administration of ZAS and OA into the trachea generated a sustained elevation of airway resistance (Raw) following IAR, while that of OA or ZAS alone resulted in Raw returning nearly to the baseline just after the IAR. The elevation of Raw after the combined challenge of OA and ZAS was significantly inhibited by pretreatment with a CysLT(1) receptor antagonist, pranlukast 30 mg/kg, but after that OA or ZAS alone was not significantly inhibited by pranlukast. The intratracheal administration of purified C5a produced an airway response that was similar to, but higher than, that evoked by ZAS. Namely, the challenge with OA plus C5a resulted in a higher IAR than OA plus ZAS, and also caused an early animal death up to 6 h, which was prevented by a combined pretreatment with pranlukast and the H(1) receptor antagonist, diphenhydramine.A histological examination at 6 h after the OA challenge identified an infiltration of inflammatory cells into the bronchial submucosal tissue, with a predominance of neutrophils and fewer eosinophils. On the other hand, a histological examination after the OA and ZAS challenge showed more severe infiltration of granulocytes into the bronchial submucosal tissue than that with OA or ZAS alone. The challenge with OA plus C5a was associated with severe perivascular leakage in the lungs and the combined pretreatment with both the antagonists led to a marked reduction in perivascular leakage. The quantitation of N-acetyl-leukotriene E(4) (N-Ac-LTE(4)), a major metabolite of cysteinyl-leukotrienes (cysLTs), in the bile indicated a significantly greater and longer excretion of cysLTs, from 1 to 6 h after the combined challenge, than that after either OA or ZAS alone. This suggested a prolonged generation of cysLTs in the lung by the combined challenge.In conclusion, our findings suggest that anaphylatoxin C5a may mediate the airway inflammatory response induced by a specific antigen challenge partly through a prolonged production of cysLTs and the release of histamine.

The pancreas as a source of cardiovascular cell activating factors.

OBJECTIVE: Physiological shock leads to elevated levels of plasma factors that activate circulating leukocytes and endothelial cells, thereby compromising microvascular functions. The nature and source of these plasma-derived activators are unknown. To examine the possible origin of these factors, we homogenized rat internal organs and measured their activity on cardiovascular cells in vivo and in vitro. METHODS: Fresh tissue samples from small intestine, spleen, heart, liver, kidney, adrenals, and pancreas were homogenized. Their ability to induce leukocyte pseudopod formation and nitroblue tetrazolium (NBT) reduction was tested and their impact in vivo on blood pressure, survival, and microvascular cell injury was examined. RESULTS: A dramatic increase (p < 0.001) in leukocyte activation compared to controls was observed with pancreas homogenate but not with homogenates from the other organs. Leukocyte activation was induced by homogenates of other tissues only after prior incubation with substimulatory concentrations of pancreatic homogenate. Pancreatic serine proteases, trypsin and chymotrypsin, which did not stimulate leukocytes, also generated activity from other tissues. Leukocyte pseudopod formation could be significantly inhibited by adding the serine protease inhibitor 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (ANGD) during tissue homogenization (p < 0.001). Injection of pancreatic homogenate into rats led to increased plasma hydrogen peroxide levels and an instantaneous drop in mean arterial pressure that was often lethal. These responses were prevented by prior infusion of ANGD (p < 0.001). Intravital microscopy of the rat mesentery confirmed that superfusion of filtered pancreatic homogenate leads to significant increases in cell death (p < 0.05), as detected by propidium iodide, and hydrogen peroxide formation (p < 0.05), as determined by dichlorofluorescein diacetate (DCFH) fluorescence. CONCLUSION: These results suggest that pancreatic enzymes attack tissue and generate cellular activators that are associated with organ dysfunction in shock.

Plasma activation during splanchnic arterial occlusion shock.

During circulatory shock, activating factors for cells in the microcirculation can be detected in plasma. But the source of such activators has remained uncertain. We have demonstrated recently that homogenates derived from the pancreas but not other peritoneal organs activate naive leukocytes. Production of such activating factors can be blocked by a serine protease inhibitor. Thus, factors generated by pancreatic proteases may possibly produce cellular activation in vivo. Rats were subjected to 90 min of superior mesenteric and celiac artery occlusion followed by reperfusion (SAO shock). In addition, rats were subjected to SAO shock for 120 min, after a 60-min pretreatment prior to occlusion with either saline or the serine protease inhibitor Futhan (nafamostat mesilate, 3.3 mg/kg b.w.). A sham SAO protocol was carried out as a control. Cellular activation was tested by neutrophil pseudopod formation and NBT reduction. Plasma from SAO-shocked animals but not sham shock rats exhibited a significant increase (P < 0.001) in the activation of naive leukocytes. Futhan-treated animals subjected to SAO shock exhibited a significantly higher post-reperfusion blood pressure than non-treated animals (P < 0.005 for all time points greater than 120 minutes), as well as significantly greater survival (P < 0.001). Neutrophil pseudopod formation and plasma peroxide production, an additional index of cellular activation, were significantly lower in Futhan-treated SAO shock plasma (P < 0.05) than levels in non-treated SAO shock animals. These results demonstrate that activating factors for leukocyte are released in SAO shock and can be mitigated by pretreatment with the serine protease inhibitor Futhan. Proteolytically derived plasma factors released during SAO shock may contribute to leukocyte activation and ensuing organ dysfunction.

Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment.

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR-/-) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-gamma mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR-/- macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1alpha and -1beta, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR-/- mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.

Mixed effects of TGF-beta on human airway epithelial-cell chemokine responses.

We investigated chemokine responses of human airway epithelial cells to transforming growth factor (TGF)-beta alone and in combination with tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. TGF-beta selectively induced production of granulocyte-macrophage colony stimulating factor (GM-CSF) without significant coordinate expression of IL-8 or RANTES. TNF-alpha induced expression of both IL-8 and GM-CSF, without detectable production of RANTES. TGF-beta synergistically enhanced GM-CSF production with TNF-alpha, but suppressed production and release of IL-8. IFN-gamma induced RANTES production and release; TGF-beta synergistically enhanced RANTES release induced by IFN-gamma, but had no effect on RANTES mRNA production. Taken together, these data demonstrate that TGF-beta may play a pivotal role in the responsiveness of airway epithelial cells to chemotactic cytokines, by selectively enhancing GM-CSF and RANTES production while suppressing IL-8 production. This profile of chemokine responses promoted by TGF-beta would favor eosinophil, lymphocyte and monocyte recruitment, hallmarks of chronic and allergic inflammation, over neutrophil sequestration.

C3a and C5a enhance granulocyte adhesion to endothelial and epithelial cell monolayers: epithelial and endothelial priming is required for C3a-induced eosinophil adhesion.

Effects of the anaphylatoxins C3a and C5a on eosinophil and neutrophil adhesion to HUVEC and to primary culture human bronchial epithelial cells (HBEC) were investigated. Activities on both leukocytes and on structural cells were examined. C3a upregulated beta2 integrin expression and caused shedding of L-selectin on eosinophils, but had no effect on neutrophil adhesion molecule expression. C5a upregulated beta2 integrins and caused shedding of L-selectin on both eosinophils and neutrophils. The potency of C5a was equivalent on both cell types; however, the magnitude of the changes in each of these adhesion molecules was significantly greater in neutrophils than eosinophils. Neither C3a nor C5a altered expression of ICAM-1, VCAM-1, E-selectin or P-selectin on either HUVEC or HBEC. C5a induced adhesion of both neutrophils and eosinophils to unstimulated HUVEC or HBEC, and adhesion was further enhanced when HUVEC and HBEC were "primed" with TNF-alpha and IFN-gamma, respectively. C3a failed to enhance adhesion of either eosinophils or neutrophils to unprimed HUVEC or HBEC, and enhanced only eosinophil adhesion to cytokine-primed HUVEC or HBEC. Similar to C3a, C3a(desArg) and a C3a-analog peptide E7 also enhanced eosinophil adhesion only to cytokine-primed HUVEC and HBEC. These results support the traditional view of anaphylatoxins as leukocyte-specific mediators. The specificity of C3a for eosinophils implicates this molecule as a potential participant in allergic inflammation. The pro-adhesive effects of C3a(desArg) suggest that this molecule, previously characterized as a spasmogenically inactive derivative of C3a, may also alter leukocyte dynamics and migration. Finally, activation of endothelium may represent an important control mechanism for C3a-mediated adhesion preventing unchecked eosinophil adhesion to uninflamed systemic vasculature.

Plasma C3a and C4a levels in liver transplant recipients: a longitudinal study.

Liver transplant patients were enrolled in a study designed to investigate correlations between plasma complement C3a or C4a levels and various postoperative complications. Longitudinal EDTA-plasma levels of C3a and C4a were measured by quantitative radioimmunoassay. Acute rejection gave a characteristic and marked increase in blood C3a, C4a and gamma-glutamyl transferase (gammaGT) levels, which rapidly resolved after high dose steroid treatment. Cytomegalovirus (CMV) infections in two of three patients gave an initial small increase only in C3a levels (i.e., alternative pathway activation) followed approximately 6 weeks later by a marked increase in C4a levels (i.e., classical or lectin pathway activation). In a third patient diagnosed for CMV infection, the complement activation profile was complicated by a coincident minor rejection episode. However, a late stage elevation in C4a was also noted. Two patients experiencing biopsy proven recurrent hepatitis C infections following transplantation exhibited increases in both gammaGT and C4a levels, without a significant increase in the level of C3a. Several hepatitis C and one hepatitis B patient had multiple late activation episodes involving marked elevation in both plasma C3a and C4a levels without detectable increases in the liver enzymes conventionally used to monitor organ function. We also showed that ex vivo activation of complement in EDTA plasma from all transplant patients was abnormally high. The classical or lectin pathway is believed to be responsible for this excessive ex vivo complement activation in the plasma of these patients. Therefore, subclinical rejection episodes and/or viral infections may be effectively detected or monitored by measuring C3a and C4a levels in plasma samples from liver transplant patients. Routine measurement of plasma complement products may provide an early non-invasive mode for detecting infections and also serve to monitor chronic or acute changes in the patient's immune system.

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Ligand binding sites on guinea pig C3aR: point and deletion mutations in the large extracellular loop and vicinity.

Human C3a receptor (huC3aR) belongs to the G-protein coupled receptor family chacterized by having seven transmembrane domains. The huC3aR is a unique member of this family having a large extracellular (EC) loop of 175 amino acids between the 4th and 5th transmembrane domains. Based on a comparison of C3aR sequences from several species, a number of charged and conserved amino acids (Asp182, Asp309, Asp310, and Arg331) in and near the large EC loop of guinea pig C3aR were replaced using site-directed mutagenesis. Competitive binding assays showed that changing Arg331 in guinea pig C3aR to Ala (or Gln), but not changing Asp182, Asp309, or Asp310 to Ala, resulted in complete loss of ligand binding activity. These results and major EC loop deletions demonstrated that an essential C3a binding site is present in the transmembrane portion of C3aR, but not in the large EC loop. Replacement of Arg331 by a noncharged residue was sufficient to eliminate ligand-receptor interactions.

Association of urinary leukotriene E4 excretion during aspirin challenges with severity of respiratory responses.

BACKGROUND: Asthmatics with aspirin- (ASA) sensitive respiratory disease (ASRD) have a spectrum of respiratory reactions during oral ASA challenge that vary in severity and are temporally associated with leukotriene (LT) formation. OBJECTIVE: This study investigates the relationship of the severity of ASA-induced respiratory reactions to urinary LTE(4) excretion. METHODS: Asthmatics with suspected ASRD underwent oral ASA challenges. Urinary LTE(4) levels were measured at baseline, during the reaction, and after acute ASA desensitization. RESULTS: Asthmatics who had respiratory reactions during oral ASA challenges were divided into 3 groups: asthmatics with naso-ocular reactions and <15% decline from baseline FEV(1) values (group 1), asthmatics with a decline in FEV(1) of 20% to 30% (group 2), and asthmatics with a decline in FEV(1) of >30% (group 3). There were no significant differences in age, baseline FEV(1) values, use of inhaled corticosteroids, daily prednisone doses, prednisone bursts, duration of reactions, or average provoking doses of ASA among the groups. At baseline group 3 asthmatics had significantly higher urinary LTE(4) levels than those in groups 1 or 2. At the time of respiratory reaction to ASA, the urinary LTE(4) levels rose significantly in all groups but were significantly greater among group 3 asthmatics compared with those in groups 1 and 2. CONCLUSION: The severity of the respiratory reactions during oral ASA challenges was associated with the degree of elevation of baseline LTE(4) synthesis. Our results suggest that asthmatics with ASRD have a spectrum of respiratory tract reactions in which leukotrienes play a distinguishing role.

Upper airway epithelial cells support eosinophil survival in vitro through production of GM-CSF and prostaglandin E2: regulation by glucocorticoids and TNF-alpha.

Production of GM-CSF by epithelial cells has been implicated in eosinophil survival within the airways, although GM-CSF promotes neutrophil and monocyte survival as well. Using primary cultures of human airway epithelial cells, we undertook a comprehensive examination of factors that enhance eosinophil survival or apoptosis. Unstimulated epithelial cells were compared to epithelial cells stimulated with TNF-alpha in the presence or absence of dexamethasone. A striking increase in survival was observed when peripheral blood eosinophils were cultured with supernatants derived from unstimulated and TNF-alpha-stimulated epithelial cells. Cultured epithelial cells were examined for transcripts of cytokines shown to enhance eosinophil survival (GM-CSF, IL-3, IL-5, IL-13, and IFN-gamma), and transcripts for cytokines promoting apoptosis (IL-10 and TGF-beta). GM-CSF transcripts, but not the other cytokines, were present in unstimulated epithelial cells, and levels were increased with TNF-alpha stimulation. TNF-alpha stimulation increased the levels of GM-CSF and PGE2 in epithelial cell supernatants and dexamethasone suppressed the TNF-alpha induced increases. The survival effects of the TNF-alpha-stimulated supernatants were effectively blocked by neutralizing antibodies to GM-CSF or by dexamethasone treatment of epithelial cells. Selectivity of GM-CSF for eosinophil versus neutrophil survival was demonstrated and suggests that epithelial cell regulation of GM-CSF and PGE2 contribute to eosinophil survival in vitro and may contribute to eosinophil accumulation in allergic disease.

Chemotactic mediator requirements in lung injury following skin burns in rats.

Partial-thickness skin burns have been shown to induce neutrophil-dependent microvascular injury both locally (skin) and systemically (lung). In the present study, interventional measures to block inflammatory chemoattractants were employed to define the pathophysiologic role of these mediators in the development of secondary lung injury following thermal injury of skin. Rats were treated with blocking antibodies to either C5a or to the alpha-chemokines, keratinocyte-derived cytokine (KC), or macrophage inflammatory protein-2 (MIP-2). To study the role of platelet activating factor, a receptor antagonist (PAF-Ra) was utilized. The development of lung vascular injury following thermal injury to skin was significantly attenuated by treatment with anti-C5a (84%), anti-KC (67%), and anti-MIP-2 (77%), but treatment with PAF-Ra had no protective effects. Protective interventions were paralleled by significant reductions in the tissue buildup of myeloperoxidase. When bronchoalveolar lavage fluids from thermally injured rats were evaluated, elevations in TNF;ZA and IL-1 were found and were determined to be C5a-dependent (but unaffected by treatment with PAF-Ra). These studies indicate that lung tissue injury after thermal skin burns is dependent on chemotactic mediators. The data also suggest that lung expression of TNFalpha and IL-1 after thermal injury of skin is C5a-dependent.

Role of chemotactic factors in neutrophil activation after thermal injury in rats.

Acute thermal trauma is well known to produce evidence of a "systemic inflammatory response" in vivo, as manifested by evidence of complement activation, appearance in plasma of a variety of inflammatory factors, and development of multi-organ injury. The current studies were focused on acute thermal injury of rat skin and factors responsible for accompanying activation of blood neutrophils. Acute thermal injury of rat skin resulted in a time-dependent loss of L-selectin and up-regulation of Mac-1 (CD11b/CD18) on blood neutrophils, with no changes in LFA-1 (CD11a/CD18). The loss of L-selectin was prevented by blockade of C5a but not by blockade of the alpha-chemokine, macrophage inflammatory protein-2 (MIP-2). C5a, the alpha chemokines, MIP-2 and keratinocyte-derived cytokine (KC), and platelet activating factor (PAF) contributed to up-regulation of blood neutrophil Mac-1. Blocking interventions against these mediators also blunted the degree of neutropenia developing after thermal trauma. These data suggest that activation of blood neutrophils after thermal trauma is related to the role of several chemotactic mediators. These studies may provide clues regarding factors responsible for development of the "systemic inflammatory response syndrome" after thermal injury in the experimental model employed.

Possible mechanism for in vitro complement activation in blood and plasma samples: futhan/EDTA controls in vitro complement activation.

BACKGROUND: Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. METHODS: Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. RESULTS: In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. CONCLUSION: The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.

Polymorphonuclear leukocytes (PMN) and eosinophils (Eos) are important cellular participants in a variety of acute and chronic inflammatory reactions in the airway. Histologic evidence has implicated direct interactions between these two subsets of leukocytes and airway epithelial cells during inflammation. A comprehensive characterization and comparison of physiologic stimuli and adhesion molecule involvement in granulocyte-epithelial-cell interactions done with nontransformed human airway epithelial cells has not been reported. We therefore examined the regulation and biochemical mechanisms governing granulocyte-epithelial-cell adhesion, using either purified PMN or Eos and primary cultures of human bronchial epithelial cells (HBECs). We investigated the involvement of a number of proinflammatory signals associated with allergic and nonallergic airway inflammation, as well as the contribution of several epithelial and leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and members of the beta(1), beta(2), and beta(7) integrin families. ICAM-1 was expressed at low levels on cultured HBECs and was markedly upregulated after stimulation with interferon (IFN)-gamma or, to a lesser extent, with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1. VCAM-1 was not present on resting HBECs, and was not upregulated after stimulation with IFN-gamma, IL-1, IL-4, or TNF-alpha. PMN adhesion to HBECs could be induced either through activation of PMN with IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), or C5a, but not with IL-5 or by preactivation of HBECs with TNF-alpha or IFN-gamma. Blocking antibody studies indicated that PMN-HBEC adherence depended on beta(2) integrins, primarily alpha(M)beta(2) (Mac-1). Adherence of Eos to HBECs could be induced through activation of Eos with IL-5, GM-CSF, or C5a, but not with IL-8 or by prior activation of HBECs with TNF-alpha of IFN-gamma. Maximal adhesion of Eos and PMN required pretreatment of HBECs with either TNF-alpha or IFN-gamma in addition to leukocyte activation. Adherence of Eos to unstimulated HBECs was mediated through both beta(1) and beta(2) integrins, whereas adhesion of Eos to activated HBECs was dominated by beta(2) integrins. Adhesion of both Eos and PMN was inhibited by treatment of HBECs with blocking antibodies to ICAM-1. Differential utilization of beta(1) and beta(2) integrins by Eos, depending on the activation state of the epithelium, is a novel finding and may affect activation and/or recruitment of Eos in airway tissue. Mechanisms of adhesion of HBECs to Eos and PMN, as evidenced by the different responsiveness of the two latter types of cells to IL-8 and IL-5, may account for a prevalence of Eos over PMN in certain airway diseases.

Multiple epithelial cell-derived factors enhance neutrophil survival. Regulation by glucocorticoids and tumor necrosis factor-alpha.

We examined the potential of several epithelial-derived factors to enhance neutrophil activation and survival. Neutrophils incubated in the presence of supernatants from nasal-derived primary epithelial cultures had significantly increased survival compared with neutrophils cultured in media alone. Of the cytokines reported to enhance neutrophil survival, transcripts for interleukin (IL)-1alpha, IL-1beta, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF) (but not interferon-gamma or granulocyte colony-stimulating factor [G-CSF]) were detected by ribonuclease protection assay in basal and tumor necrosis factor (TNF)-alpha- stimulated epithelial cells. Of the eicosanoid products that enhance neutrophil survival, platelet-activating factor and leukotriene B(4) were not detected in the supernatants, whereas prostaglandin E(2) (PGE(2)) was produced in modest amounts. The levels of IL-6, GM-CSF, and PGE(2) in epithelial supernatants were significantly increased after transient TNF-alpha stimulation. This induction was suppressed if dexamethasone (Dex) was added during TNF-alpha stimulation. Only IL-6, GM-CSF, and PGE(2) promoted neutrophil survival over the range of concentrations detected in the supernatants, and a combination of neutralizing antibodies to GM-CSF and IL-6 completely inhibited the enhanced neutrophil survival in epithelial supernatants. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and morphologic scoring of apoptotic neutrophils confirmed that epithelial supernatants, as well as purified IL-6, GM-CSF, and PGE(2) all delayed neutrophil apoptosis. Finally, the effects of Dex on neutrophil survival and on epithelial cytokine production were investigated. Dex independently prolonged neutrophil survival but suppressed epithelial production of survival-enhancing factors in a dose-dependent manner. The net effect of Dex appeared to favor neutrophil survival.

Role of the second extracellular loop of human C3a receptor in agonist binding and receptor function.

The C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor with an unusually large second extracellular loop (e2 loop, approximately 172 amino acids). To determine the function of this unique structure, chimeric and deletion mutants were prepared and analyzed in transfected RBL-2H3 cells. Whereas replacement of the C3aR N-terminal segment with that from the human C5a receptor had minimal effect on C3a binding, substitution of the e2 loop with a smaller e2 loop from the C5a receptor (C5aR) abolished binding of 125I-C3a and C3a-stimulated calcium mobilization. However, as much as 65% of the e2 loop sequence (amino acids 198-308) may be removed without affecting C3a binding or calcium responses. The e2 loop sequences adjacent to the transmembrane domains contain multiple aspartate residues and are found to play an important role in C3a binding based on deletion mutagenesis. Replacement of five aspartate residues in the e2 loop with lysyl residues significantly compromised both the binding and functional capabilities of the C3a receptor mediated by intact C3a or by two C3a analog peptides. These data suggest a two-site C3a-C3aR interaction model similar to that established for C5a/C5aR. The anionic residues near the N and C termini of the C3aR e2 loop constitute a non-effector secondary interaction site with cationic residues in the C-terminal helical region of C3a, whereas the C3a C-terminal sequence LGLAR engages the primary effector site in C3aR.