Stimulation of rat hepatocyte fibronectin production by monocyte-conditioned medium is due to interleukin 6.

In this report, conditioned media from LPS-stimulated monocytes increased rat hepatocyte production of fibronectin (Fn) in a dose-dependent manner. Preincubation of the conditioned media with anti-IL-6, but not with anti-IL-1 alpha, anti-IL-1 beta, or anti-TNF, completely neutralized the Fn-stimulating activity. 10-100 pg/ml of rIL-6 was sufficient to increase Fn production. Neither IL-1 nor TNF had an effect on Fn production. The Fn-stimulating activity of IL-6 could be specifically neutralized only with anti-IL-6, but not with anti-IL-1 or anti-TNF. The increased Fn produced was shown to be of the plasma rather than the cellular form. These results demonstrate that IL-6 is the factor in monocyte-conditioned media that stimulates Fn production, and that IL-6 is the monokine tested with such activity.

Development of the brachial lateral motor column in the wingless mutant chick embryo: motoneuron survival under varying degrees of peripheral load.

Survival of motoneurons in the lateral motor column (LMC) of the chick embryo is known to depend on the periphery. How this dependence relates to the normally occurring death of motoneurons is unknown. Analysis of the time course of LMC cell loss in the absence of varying amounts of limb musculature could help bring about an understanding of this relationship. We undertook this analysis by studying LMC development in wingless chick embryos. Grossly these embryos lack wings, but we have reported that some of them possess more than 40% of the normal volume of wing bud-derived muscles (M.E. Lanser and J.F. Fallon, Anat. Rec. 217:61-78, 1987). In the present work we compared the time course of LMC development in wingless embryos that possessed varying amounts of wing bud-derived musculature with that in normal embryos. In normal embryos little cell loss occurs from the brachial LMC prior to day 8 (15% of the total cell loss). Most of the normal cell loss occurs between 8 and 10 days (62% of the total cell loss). In the wingless LMC, anywhere from 55% to 70% of the total cell loss occurs before day 8. The death of motoneurons prior to day 8 is proportional to the amount of wing bud musculature eliminated by the mutation. Cell loss after day 8 is proportional to the amount of wing bud musculature spared by the mutation. Therefore, when the limb is missing, most motoneurons die before the major period of cell loss even begins in the normal LMC. Counts of dead cells in the LMC also support this conclusion. In addition, curves plotting the rates at which cells are lost from the brachial LMC provide a suggestion that normal cell loss is biphasic and that limb removal enhances primarily the first phase of cell loss. These data suggest that the majority of motoneurons may die for different reasons in the normal and the limb-deprived LMCs. Overall, the number of motoneurons surviving in the brachial LMC is proportional to the volume of wing bud-derived muscle present. However, as the muscle volume approaches zero, motoneuron number does not. This suggests that most, but not all, motoneurons depend on limb bud-derived muscles for survival. Finally, the decreased motoneuron number in the wingless LMC, when compared to normal after the cell death period, cannot be totally accounted for by the additional loss of cells that occurred during the cell death period in the wingless LMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutrophil-mediated lung localization of bacteria: a mechanism for pulmonary injury.

The reticuloendothelial system (RES) is thought to ensure organ integrity following trauma, burn, and sepsis by removing potentially embolic particulate matter and blood-borne bacteria from the circulation. Blockade of the RES with foreign colloids is known to result in a consumptive depletion of opsonic fibronectin, which modulates reticuloendothelial function, and an increase in lung localization of test particles. We investigated the role of neutrophils as a contributing factor in the increased localization of blood-borne bacteria in the lung after blockade. RE blockade induced by gelatin-coated colloid particle injection resulted in an acute (15-minute) increase in the number of 51Cr-labeled neutrophils localized in the lung, with return to control levels at 60 minutes after blockade. Fibronectin administration following blockade resulted in a significant (P less than 0.05) prolonged retention of neutrophils in the lung up to 2 hours after blockade. A parallel increase (P less than 0.05) in lung localization of heat-killed 14C-labeled Pseudomonas aeruginosa following colloid-induced RE blockade was observed, and fibronectin further increased the number of bacteria localized in the lung. Experimentally induced neutropenia abrogated the effect of colloid injection on lung localization of bacteria. It is concluded that a particulate load results in simultaneous RE blockade and neutrophil margination in the lung, both of which contribute to the increase in lung localization of bacteria. A mechanism for neutrophil-mediated pulmonary injury related to RE dysfunction following trauma is proposed.

Neutrophil CR3 induction by platelet supernatants is due to platelet-derived growth factor.

Recently, serum was shown to contain a factor that increased expression of the complement receptor CR3 on neutrophil membranes. This factor was localized to platelet granules and was released during coagulation. This study was undertaken to identify this factor in platelet granules. Platelet supernatants containing granule contents were incubated with neutrophils, and CR3 expression was determined by flow cytometry. Incubation with platelet supernatants induced more than a twofold increase in the amount of CR3 expressed on the neutrophil membrane (p = 0.05). Anti-platelet-derived growth factor (anti-PDGF) Fab, when preincubated with the platelet supernatants, completely inhibited this CR3-inducing activity. Pure PDGF induced a dose-response increase in CR3, whereas platelet factor 4 had no effect. PDGF was active in concentrations well within the physiologic range. These data indicate that PDGF is responsible for the CR3-inducing activity of platelet supernatants. PDGF may well be an important regulator of neutrophil adherence and phagocytic function in areas of tissue injury.

Correction of serum opsonic defects after burn and sepsis by opsonic fibronectin administration.

Opsonic fibronectin modulates reticuloendothelial (RE) uptake of nonbacterial particulates, as well as some bacterial strains, and may thus play an important role in host defense against sepsis after burn injury. We evaluated the relationship between burn injury, sepsis, and opsonic fibronectin levels in rats, as well as the ability to reverse the acute opsonic deficiency after burn injury by administration of purified opsonic fibronectin. Burn injury resulted in an acute (within one hour) depletion of opsonic fibronectin (from 341 +/- 30 to 98 +/- 7 mg/L) that was correctable by administration of purified opsonic fibronectin when accompanied by moderate sepsis, while burn injury plus severe sepsis (level, 168 +/- 30 mg/L) limited attempted restoration of normal opsonic levels (level, 121 +/- 18 mg/L). The in vitro serum opsonic deficit was partially correctable (from 2.2% to 6.7% of the injected dose per 100 mg), while in vivo RE functional deficits could not be corrected. We conclude that the acute postburn deficiency in opsonic fibronectin is amenable to repletion therapy; however, many additional factors may contribute to acute RE failure after burn injury.

Coagulation augments neutrophil C3b receptors via formation of a protein(s) unrelated to fibrinolysis or C5 activation.

The present study investigated the effect of coagulation on neutrophil complement receptors (CRs) 1 and 3, which are specific for the opsonins C3b and C3bi. Incubation of neutrophils in autologous serum, but not in plasma, increased the mean (+/- SD) expression of CR1 (x3.43 +/- 0.93) and CR3 (x3.07 +/- 0.86), in comparison with incubation in buffer. Serum also increased neutrophil superoxide production in response to opsonized zymosan from 0.48 +/- 0.21 to 1.05 +/- 0.25 nmol/10(6) cells/min. Similarly, calcium conversion of platelet-rich plasma (but not platelet-poor plasma) to serum also increased both CR1 and CR3 expression. This finding, as well as the fact that freeze-thawed platelet-rich plasma (but not platelet-poor plasma) increased CR expression, indicated that platelet constituents were the origin of this CR-inducing activity. Other nonplatelet factors formed during coagulation, such as C5a, fibrinogen degradation products, kallikrein, and factor XIIa, were shown not to be responsible for this CR-inducing activity.

Trauma serum suppresses superoxide production by normal neutrophils.

The effect of trauma serum on superoxide production by normal neutrophils was studied in 47 serum samples from 18 patients with multiple trauma. Ten patients became septic and eight patients remained nonseptic. Incubation in trauma serum significantly suppressed superoxide production by normal neutrophils compared with incubation in normal serum: 3.6 +/- 1.44 vs 4.04 +/- 1.64 nmole of superoxide produced by 10(6) neutrophils (mean +/- SD). There was no difference in the suppressive effect between septic and nonseptic trauma serum samples. The chemiluminescence response of normal neutrophils was likewise suppressed following incubation in trauma serum compared with incubation in normal serum. The chemiluminescence response correlated with superoxide reduction of cytochrome C. In addition, the chemiluminescence response was significantly less in septic-trauma serum than in nonseptic-trauma serum. Suppressive serum was found to inhibit the neutrophil-membrane depolarization response to latex particles, as measured by flow cytometry. We conclude that trauma serum suppresses superoxide production by normal neutrophils, and that such suppression can be detected reliably using the clinically applicable technique of chemiluminescence. A normal chemiluminescence response excludes serum-mediated suppression of neutrophil superoxide production. In addition, chemiluminescence may be of value in detecting altered resistance to sepsis following injury, while superoxide determinations do not seem to be helpful in this regard. The mechanism of action of the suppressor may involve reversible inhibition of membrane depolarization necessary for the production of bactericidal oxygen species.

Analysis of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane in rat plasma. II. Pharmacokinetic profile in male and female Sprague-Dawley rats evaluated by capillary electrophoresis.

This paper describes a pharmacokinetic study performed in Sprague-Dawley rats after i.v. administration of a single 6-mg/kg dose of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane (Altropane). Plasma samples were collected from the retro-orbital sinus at times up to 3 h after drug administration, extracted by solid-phase extraction, and the drug levels determined by capillary electrophoresis (CE). Pharmacokinetic parameters were determined by a standard noncompartmental model using WinNonlin version 1.5. The maximum plasma concentrations, clearances of the drug, and areas under the curve for male and female rats were 5.74 and 7.26 microg/ml, 135.7 and 98.5 ml/kg x min, and 44.23 and 60.92 microg x min/ml, respectively. The drug was cleared very rapidly from the systemic circulation, with a terminal t(1/2) of 7 to 10 min and a mean residence time of about 11 min for both sexes. The volume of distribution was approximately 1 l/kg. No metabolites were detected when the samples were analyzed individually. However, after samples were pooled and concentrated, traces of two unknown peaks that may represent metabolites were detected in concentrates from the last two timepoints. Part I of this work [J. Chromatogr. A, 895 (2000) 87] describes validation of CE methods for the analysis of aqueous and plasma samples of Altropane, including its solid-phase extraction from rat plasma.

Amiprilose hydrochloride for the treatment of rheumatoid arthritis.

The intent of this study was to compare, in a monotherapy framework, an optimal dose of the synthetic hexose sugar, amiprilose hydrochloride (HCl), to a placebo in the treatment of rheumatoid arthritis. In this double-blind, randomized, multi-center study, patients first underwent a washout period from disease-modifying antirheumatic drugs. Those who subsequently met flare criteria within 14 days of discontinuing previously stable doses of nonsteroidal anti-inflammatory drugs were randomized to amiprilose HCl (103 patients) or a placebo (115 patients) for the subsequent 20 weeks. Glucocorticoid or nonsteroidal anti-inflammatory drugs use was not permitted. At the baseline, demographic and disease characteristics were similar in both groups. Of patients completing the course of therapy, 73% were in the amiprilose HCl group and 66% were in the placebo group. Using an intent-to-treat analysis, numeric trends favoring amiprilose HCl treatment were found for clinical and laboratory parameters of disease activity. Compared with the placebo group, statistically significant degrees of improvement were achieved for the number of swollen joints (p /= \50% reduction in swollen joints (p

Induction of hepatocyte synthesis of fibronectin by a non-interleukin-1 monokine.

Plasma fibronectin concentrations initially decrease and then markedly increase following injury and during inflammation. Although hepatocytes are known to be the major source of plasma fibronectin, the stimulus controlling its production is not known. The present study investigated the role of interleukin-1 (Il-1) and LPS-induced monocyte supernatants in stimulating hepatocyte fibronectin synthesis in vitro. Hepatocytes exposed to monocyte supernatants significantly increased their production of fibronectin and fibrinogen. Albumin production was suppressed. Both LPS-induced and noninduced monocyte supernatants increased hepatocyte fibronectin and fibrinogen synthesis, and decreased albumin synthesis. However, only LPS-induced monocyte supernatants contained Il-1 activity. Pure Il-1 had no effect on hepatocyte fibronectin synthesis. Molecular sizing of the monocyte supernatants containing the Fibronectin Stimulating Factor (FSF) yielded active fractions in the 30-40 kD range. These fractions did not contain Il-1 activity. We conclude that a non-Il-1 monokine, Fibronectin Stimulating Factor (FSF), stimulates hepatocyte production of fibronectin. FSF is presumably the monokine responsible for stimulating hepatocyte fibronectin production in vivo following injury and during inflammation. Based on these results, and the time-course of fibronectin levels seen following injury and during inflammation, it seems reasonable to consider fibronectin as one of the acute-phase proteins.

Development of wing-bud-derived muscles in normal and wingless chick embryos: a computer-assisted three-dimensional reconstruction study of muscle pattern formation in the absence of skeletal elements.

The mechanisms whereby the normal pattern of muscles within the developing chick limb bud is generated are largely unexplored. It has been proposed that the muscle pattern is established independently of the pattern for the limb skeletal elements to which the muscles normally attach (Shellswell and Wolpert: "The Pattern of Muscle and Tendon Development in the Chick Wing."In: Vertebrate Limb and Somite Morphogenesis. Cambridge University Press, Cambridge, pp. 71-86, 1977). To further examine this possibility we studied the formation of the proximal wing muscles in normal and wingless chick embryos. The muscles of the shoulder region (including the pectoralis) arise as part of the dorsal and ventral premuscle masses of the developing limb bud. These secondarily migrate out of the limb to take origin from the pectoral girdle while inserting onto the humerus (Sullivan: Aust. J. Zool., 10:458-516, 1962). With rare exceptions, wingless embryos have complete absence of wing skeletal elements, but they may possess more than 40% of the normal volume of wing-bud-derived muscles. The muscles that remain in wingless embryos are primarily shoulder muscles, and to a varying extent, the pectoralis. The question we sought to answer was whether in wingless embryos the proximal wing muscles could form a normal pattern in the absence of the humerus and distal wing skeletal elements. By examining three-dimensional reconstructions of the proximal wing region in normal and wingless embryos, we found that the initial subdivision of the dorsal and ventral premuscle masses proceeded normally in the absence of the wing skeleton. This resulted in a grossly normal pattern of proximal wing muscles despite the absence of wing skeletal elements. However, some subsequent cleavages of individual muscles within premuscle mass divisions did not occur in wingless embryos. This suggests that the skeleton may be required for this step in muscle morphogenesis to occur. We also observed that the wing-bud-derived muscles in wingless embryos were nearly always anchored to the pectoral girdle at both ends. Sometimes this resulted in muscles making abnormal tendonous fusions with other muscles derived from the opposite (i.e., dorsal or ventral) premuscle mass. Therefore, attachment to the skeleton may be important for some facet of muscle development. Finally, the supracoracoideus muscle was absent in all but one wingless embryo we examined in the present study. In that one, it was substantially reduced in volume compared to normal. absence of this muscle, the space normally occupied by the supracoracoideus was maintained beneath the pectoralis.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of the lateral motor column in the limbless mutant chick embryo.

This is a report on the development of the lateral motor column (LMC) in the limbless mutant chick embryo. The limbless mutant was used to study the effects of the absence of a periphery on the developing nervous system. The limbless mutant provides a unique opportunity to compare the effects on the LMC of deletion of a limb caused by the genotype with those seen following surgical removal of the limb primordium. Cell counts of the total number of motoneurons in the LMC at both the brachial and lumbar levels were done in a large series of limbless embryos and on their normal siblings. In normal embryos, a substantial loss of LMC motoneurons was observed during the course of normal development. At the brachial level, 54% of the initial LMC cell population was lost between day 6 and day 18. At the lumbar level, 40% of the initial population was lost between the 6th and 12th days of development with no further loss through day 18. An even more massive cell loss was observed in the limbless mutant LMC at both brachial and lumbar spinal cord levels between day 5 and day 12. This resulted in the elimination of at least 85% of the motoneurons that were initially present in the limbless LMC. Our data demonstrate that the effects of peripheral deprivation on LMC development in the limbless mutant are similar to those seen following surgical removal of the periphery. The initial production of motoneurons and assembly of the LMC did not appear to be significantly affected by the mutation, while the subsequent degeneration of LMC motoneurons is greatly accelerated and increased in comparison to the normal.

Opsonic fibronectin is necessary for optimal serum-mediated phagocytosis of Staphylococcus aureus by human neutrophils.

Opsonic fibronectin is known to mediate reticuloendothelial (RE) cell and neutrophil uptake of nonbacterial particulates. In a recent study opsonic fibronectin deficiency following burn preceded the onset of sepsis, leading us to hypothesize a role for this protein in antibacterial defense. To test this hypothesis we compared pooled normal human serum to fibronectin-depleted serum in its ability to opsonize and promote phagocytosis of Staphylococcus aureus by human neutrophil monolayers. Phagocytosis and intracellular killing were evaluated using acridine orange staining and ultraviolet (UV) microscopy. Human serum depleted of opsonic fibronectin by gelatin-sepharose affinity chromatography manifested a marked reduction in its ability to support phagocytosis of S aureus by human neutrophils. Reconstitution of fibronectin-deficient human serum with purified human plasma fibronectin restored its opsonic activity. The direct interaction of fibronectin with the bacteria was shown by mixing and/or incubation of the bacteria with normal serum followed by centrifugation and removal of the bacteria. This resulted in a marked (P less than 0.05) depletion (adsorption) of the fibronectin from the serum. Fibronectin appears not to act independently, but was an important cofactor in the ability for serum to stimulate phagocytosis. Thus, plasma fibronectin may be an important protein essential for maximal opsonic activity of serum. Its depletion following trauma and burn may undermine RE cell and neutrophil defense against infection and bacteremia, thus contributing to organ failure during septic shock.

Opsonic fibronectin deficiency and sepsis. Cause or effect?

Opsonic fibronectin is known to modulate macrophage (RE cell) and neutrophil Phagocytic function. Its depletion has been documented following trauma, burn, and operation in patients with rapid restoration of normal levels unless bacteremia and/or wound sepsis intervenes. Sepsis is associated with a secondary phase of opsonic fibronectin deficiency. We have observed in burn patients that this secondary phase of opsonic fibronectin depletion following trauma and burn is seen two to three days prior to the onset of clinical sepsis, raising the question of whether this deficiency sensitized the host to the subsequent development of sepsis or whether its deplection was merely an unsuspected sensitive indication of preclinical sepsis. To address the possibility that opsonic fibronectin deficiency might lower resistance to sepsis, Sprague-Dawley rats (200 gm) were partially depleted (35%) of their opsonic fibronectin prior to intraperitoneal inoculation with Staphylococcus aureus. Mortality to S. aureus peritonitis was significantly (p < 0.05) increased in animals with fibronectin deficiency. Furthermore, in control animals, nonsurvival was also associated with significantly (p < 0.05) lower initial fibronectin levels than survival. However, peritonitis itself also resulted in an early (within one hour) depletion of opsonic fibronectin followed by a marked "hyperopsonemia" within 12 hours in both groups. Thus, opsonic fibronectin depletion decreases resistance to sepsis, and the development of sepsis itself will initiate opsonic fibronectin deficiency. Host defense against infection may depend on early restoration and maintenance of normal opsonic fibronectin levels following trauma, burn, and operation, as well as the ability of the host to mount an appropriate hyperopsonemic elevation of fibronectin levels in response to infection.

Coagulation increases neutrophil CR1 and CR3 expression: primary role for platelet-derived growth factor.

Neutrophil receptors for C3b(CR1) and C3bi(CR3) mediate a number of functions important for infection control and tissue repair, such as adherence, aggregation, orientation in chemotactic gradients, and phagocytosis of opsonized particles. We studied the effect of the coagulation of whole blood on the induction of neutrophil complement receptor (CR) expression in vitro. Neutrophils incubated in serum for 1 hour at 37 degrees C increased the expression of CR1 3.43-fold and CR3 3.06-fold compared with incubation in buffer (p less than 0.001). In contrast, incubation in plasma did not induce such an increase. The serum factor responsible for this CR-inducing effect appeared to be a platelet constituent, because (1) serum derived from platelet-rich plasma, but not platelet-poor plasma, contained the CR-inducing factor; (2) pretreatment with aspirin inhibited the adenosine diphosphate-induced expression of this factor in platelet-rich plasma; (3) the CR-inducing factor was also contained in supernatants derived from frozen/thawed platelets; (4) pure platelet-derived growth factor (PDGF) induced CR expression to the same extent as did whole serum; and (5) the CR-inducing activity of serum and platelet supernatants was inhibited by incubation with antibody against PDGF but not by antibody against C5. Thus, a platelet component that is probably PDGF appears to be the major CR-inducing factor generated during in vitro coagulation and may play a vital role in mediating the neutrophil response to tissue injury and inflammation.

Survival of motoneurons in the brachial lateral motor column of limbless mutant chick embryos depends on the periphery.

Motoneuron survival in the embryonic spinal cord is influenced by the presence or absence of the developing limb bud. We have recently begun a reexamination of the relationship between limb absence and motoneuron survival in a nonsurgical limb deletion model, the limbless mutant chick embryo. As in surgically limb-deleted normal embryos, only 10% of the motoneurons that are initially produced in the limbless mutant lateral motor column (LMC) survive the embryonic period (Lanser and Fallon, 1984). We now report that, when supplied with a normal periphery (i.e., a normal limb bud), more than 40% of the motoneurons initially produced in the limbless LMC survive the embryonic period. Motoneuron cell counts in one-winged limbless embryos reveal that over 3.5 times as many motoneurons survive the cell death period in the LMC on the side with the limb than on the opposite, limbless side. This demonstrates the dependence of embryonic LMC motoneurons on the developing limb for survival and indicates that the limbless mutant is an appropriate model for studying the death and survival of LMC motoneurons during development. Using the limbless mutant to study LMC motoneuron survival eliminates the complication of possible direct surgical effects on motoneuron death. In addition, we found that a substantial effect of the wing on rescuing LMC motoneurons was exerted prior to the 6th day of embryonic development. Normally, little cell loss occurs in the brachial LMC during this time. Accordingly, motoneuron death in the limb-deprived brachial LMC, whether in surgically limb-deleted normal embryos or in genetically limbless embryos, is accelerated with respect to cell death in the normal brachial LMC.

Opsonic glycoprotein (plasma fibronectin) levels after burn injury. Relationship to extent of burn and development of sepsis.

The time course of immunoreactive and bioassayable opsonic alpha 2-SB glycoprotein (plasma fibronectin), as well as its relationship to both the extent of injury and development of postburn sepsis, was evaluated following burn injury. Immunoreactive opsonic fibronectin was depleted acutely within hours following burn; its maximal depletion occurring 12 hours postburn injury. The magnitude of depletion was correlated with the body surface area burned, and normal levels were restored at 24 hours postinjury. There was a tendency toward rebound hyperopsonemia at two weeks postburn, with a slow return to normal over the ensuing weeks. Bioassayable opsonic protein levels, in general, paralleled those of immunoreactive protein. Following restoration of opsonic protein levels, a secondary phase of opsonic fibronectin deficiency (p equal to 0.05) developed in those burn patients that became septic. Moreover, this opsonic fibronectin deficiency actually became apparent prior to the onset of clinical sepsis, although it was maximal during sepsis. The resolution of the septic episode was associated with the return of plasma opsonic fibronectin levels to normal. The possibility that secondary deficiency in immunoreactive opsonic fibronectin may be a reliable index of impending sepsis following burn warrants further investigation.

Neutrophil CR3 expression and specific granule exocytosis are controlled by different signal transduction pathways.

Neutrophils express receptors (CR3) for the complement fragment C3bi. CR3 expression can be increased by exposure of the cells to chemotactic factors such as FMLP or to the calcium ionophore A23187. It has been suggested that CR3 moieties are stored in the membrane bounding either the secondary or the tertiary (gelatinase containing) granules. To help define the mechanisms mediating CR3 up-regulation, the effects of several inhibitors upon CR3 expression and secondary granule exocytosis were investigated. Pertussis toxin inhibited FMLP-induced (but not A23187-induced) CR3 expression and exocytosis, indicating that an early step in FMLP-induced CR3 expression is activation of a pertussis toxin-sensitive G protein. However, CR3 expression and exocytosis appeared to be controlled by separate mechanisms distal to G protein activation because 1) DBcAMP and the protein kinase inhibitor H-7 inhibited or stimulated exocytosis, respectively, without affecting CR3 expression; 2) the calmodulin (chlorpromazine and trifluoperazine) and myosin L chain kinase (ML9) inhibitors had greater effects on exocytosis than on CR3 expression; and 3) the kinetics of CR3 expression and exocytosis differed markedly. Thus, although G protein activation is a common early step in both processes, there is a bifurcation of the two processes distally. The mechanisms mediating CR3 up-regulation and tertiary granule exocytosis were also investigated. Extracellular Ca2+ was essential for tertiary granule exocytosis, but not for CR3 up-regulation. We conclude that because the mechanisms controlling CR3 up-regulation and exocytosis diverge soon after the binding of a chemotactic ligand to its receptor, that at least the bulk of increased CR3 expression is not simply a by-product of secondary and tertiary granule exocytosis but is the result of the mobilization of CR3 moieties from an intracellular pool of uncertain identity.

Neutrophil chemiluminescence and opsonic fibronectin levels following blunt trauma.

Trauma is known to result in depression of opsonic fibronectin levels as well as abnormalities in neutrophil function. Neutrophil oxidative metabolism, important for bactericidal activity, has not been investigated following injury. Since fibronectin has been reported to increase neutrophil chemiluminescence (CL), we examined the relationship between neutrophil oxidative metabolism (as measured by chemiluminescence) and opsonic fibronectin levels following blunt trauma. Sera from 11 nonseptic and 9 eventually septic-trauma patients were studied. Normal neutrophils incubated in septic-trauma serum had decreased CL responses compared to incubation in nonseptic serum (P less than 0.0001). This difference was apparent immediately after injury, prior to the onset of sepsis. This depression was due to the presence of a serum suppressor of neutrophil chemiluminescence and not to the absence of a serum factor. This suppressor has been partially characterized as a protein of greater than 30,000 Da. Opsonic fibronectin levels were also depressed in septic-trauma sera compared to nonseptic-trauma sera (P less than 0.0001). However, no correlation could be demonstrated between the CL response and opsonic fibronectin levels. Addition of fibronectin to buffer increased the CL response, while addition of fibronectin to nonseptic-trauma serum had no effect. In contrast, addition of fibronectin to septic-trauma sera actually decreased the CL response (P less than 0.05), perhaps by forming complexes with abnormal proteins and interfering with membrane-particle interaction.