Procoagulant Activity Response Research Articles

Meconium stimulates a pro-inflammatory response in peritoneal macrophages: Implications for meconium peritonitis

Background/Purpose: Although meconium peritonitis is a rare condition, the mortality rate can be as high as 40%. Meconium peritonitis is a result of intestinal perforation in utero, which leads to dense inflammation in the peritoneal cavity. The fetus has relatively immature peritoneal defense mechanisms, so the cause of this dense inflammation is unclear. The peritoneal macrophage is a key cell in the peritoneal inflammatory response in adults. The purpose of this investigation was to determine if sterile meconium had a direct stimulatory effect on the peritoneal macrophage. Methods: Peritoneal macrophages were harvested from adult C3H HEN mice. The cells were placed in microtiter wells at 10 5 cells per well. Sterile human meconium was diluted in media and placed in the wells at varying concentrations for 8 hours. Lipopolysaccharide (LPS) (10 μg/mL) served as a positive control. Supernatants were harvested and assayed for tumor necrosis factor alpha (TNF-α) using a commercial ELISA kit. Separate cells were assayed for TNF-α message using polymerase chain reaction (PCR). In another series of experiments, procoagulant activity (PCA) was determined on freeze-thawed cells using a two-stage amidolytic assay. To test for the role of protein kinase C (PKC) in the PCA response H7, a PKC inhibitor, was used as well. Results: Meconium stimulation resulted in a significant increase in TNF-α compared with negative controls with a peak at 0.1% meconium (121 pg/mL v 11 pg/mL, P < .05). There was a significant increase in PCA, with a 10-fold increase with 1% meconium compared with controls ( P < .05). This response was limited to less than 5% by PKC inhibition. Conclusions: Sterile meconium results in a marked proinflammatory response in the peritoneal macrophage with elevations of both PCA and TNF-α. The TNF response is likely mediated at a pretranscriptional level because there is a marked increase in TNF mRNA. These data suggest that the PCA response is regulated by a PKC mechanism similar to LPS. Stimulation of the peritoneal macrophage by meconium is a possible cause of the marked inflammation seen in meconium peritonitis.

Mouse Hepatitis Virus-3 Induced Prothrombinase ( Fgl2) Maps to Proximal Chromosome 5

Infection with mouse hepatitis virus-3 (MHV-3), a member of the coronavirus family, leads to a strain-dependent spectrum of liver disease. Mice of the BALB/cJ, C57BL/6J, and DBA strains are fully susceptible, exhibiting 100% mortality when infected with as little as 0.1 PFU of MHV-3, while A/J mice are resistant, as defined by complete survival and normal liver histology after infection with 2 X 10{sup 4} PFU. All of these strains are permissive for viral replication, suggesting that host immune factors, rather than viral cytopathology, are responsible for the observed difference in mortality. While the pathogenesis of MHV-3-induced liver disease is not fully understood, several lines of evidence indicate that local activation of the coagulation cascade prior to detectable viral replication plays an important role in liver cell injury. First, microscopy performed early in the infection of susceptible mice has shown sinusoidal thrombosis and foci of coagulation necrosis associated with varying degrees of inflammatory cell infiltration. Second, a correlation between disease severity and the induction of macrophage procoagulant activity (PCA) has been established, with susceptible mice developing an earlier and heightened PCA response relative to resistant strains. Finally, treatment of mice with a monoclonal antibody to MHV-3-induced PCA prevents the lethalitymore » associated with infection. Genetic linkage in the form of an identical strain distribution pattern was established between susceptibility to infection with MHV-3 and inducible macrophage PCA, using the set of AXB/BXA recombinant inbred strains derived from resistant (A/J) and susceptible (C57BL/6J) progenitors. 18 refs., 1 tab.« less

Procoagulant and prothrombotic responses of human endothelium to indomethacin and endotoxin in vitro. Relevance to non-steroidal anti-inflammatory drug enteropathy.

In view of the association between non-steroidal anti-inflammatory drugs (NSAIDs) and microvascular injury in the intestine, this study investigated the procoagulant changes in cultured human umbilical vein endothelial cells (HUVEC) when exposed to indomethacin either alone or in the presence of bacterial lipopolysaccharide (LPS). Confluent HUVEC cultures were cultured for 1, 6, 12, and 24 h in the presence of LPS (10 micrograms/ml) with or without indomethacin (1-100 microM). After incubation, supernatants were analysed for 6-keto-prostaglandin (PG) F1 alpha and PGE2 content, whereas cells were freeze-fractured and assayed in a one-stage clotting assay for the expression of procoagulant activity (PCA). LPS induced a significant expression of PCA at 6, 12, and 24 h, with a significantly increased production of 6-keto-PGF1 alpha, whereas the increased PGE2 production was much less pronounced. Indomethacin alone induced a time-dependent PCA response; when coincubated with LPS the PCA response was greater than that produced by either indomethacin or LPS alone. Indomethacin totally abolished the synthesis of antithrombotic eicosanoids. Indomethacin induces PCA in HUVEC and augments LPS-induced PCA, while it abolishes the antithrombotic prostanoid response in these cells. These observations may be relevant to the microvascular injury and thrombosis observed in NSAID enteropathy.

Extravascular coagulation and fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Diffuse pulmonary inflammation in interstitial lung diseases is associated with increased coagulation in the extravascular spaces of the lung. We hypothesized that conditions favoring coagulation over fibrinolysis in the lung are related to inflammation. Pulmonary coagulation and fibrinolysis were studied in two strains of mice susceptible or resistant to the development of lung inflammation in response to the mycobacterial cell wall glycolipid trehalose-6,6'-dimycolate (TDM). Susceptible animals treated with TDM intravenously develop well-organized collections of mononuclear cells in the lung parenchyma referred to as granulomas in this report. More granulomas were found in the susceptible ICR mice than in the resistant A/J mice after intravenous administration of TDM (7 +/- 1 granulomas/mm2 versus 1 +/- 0.3 granulomas/mm2, p = 0.005). Granuloma formation was associated with increased lung procoagulant activity (PCA) measured in bronchoalveolar lavage (BAL) cell lysates from susceptible mice. In contrast, TDM-resistant A/J mice challenged with TDM did not have a significant BAL cell PCA response, but expressed several-fold greater levels of lung BAL fluid plasminogen activator activity (PAA) than ICR mice. To examine the role of coagulation in the TDM pulmonary inflammatory response, susceptible C57Bl/10SnJ mice were anticoagulated by oral administration of warfarin prior to challenge of TDM; these mice developed fewer pulmonary granulomas than TDM-treated mice without warfarin treatment (2.6 +/- 0.5 granulomas/mm2 versus 6.5 +/- 0.8 granulomas/mm2, p < 0.001) but had similar BAL cell PCA and lung inflammatory changes as measured by lung weights and BAL cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

LPS-induced, but not interferon-gamma-induced procoagulant activity of suspended human macrophages is followed by a refractory state of low procoagulant expression

Monocyte-derived macrophages cultured under a variety of conditions were assessed for expression of procoagulant activity (PCA) upon induction by various triggers, using a semiautomated turbidimetric recalcification time assay in a kinetic ELISA reader. Macrophages cultured in a nonadherent (teflon) culture system and seeded in microtiter plates responded with PCA expression to lipopolysaccharide (LPS), to toxic shock-syndrom toxin-1 (TSST-1) and to surface-bound IgG, but not to surface-bound albumin, nor to interferon-γ (IFN-γ). In contrast, macrophages stimulated in teflon containers by IFN-γ showed a strong PCA response peaking around 24 hr after stimulation, but they failed to secrete tumour necrosis factor (TNF). Suspended IFN-γ-stimulated cells showed a similar response upon 2nd stimulation by LPS or IgG after adherence to microtiter plates as did nonprimed counterparts. In contrast, cells primed in suspension, then cultured in adherence secreted dramatically enhanced amounts of TNF when compared with nonprimed cells. Macrophages stimulated in suspension with LPS showed a PCA response of similar magnitude, which was accompanied by TNF secretion. PCA of both IFN-γ-primed and LPS-exposed suspension culture cells was largely due to the surface expression of tissue factor, and to a lesser extent of a prothrombinase-like activity, as evidenced by PCA testing with factor-X-deficient plasma. The kinetics of LPS-induced PCA differed from IFN-γ-induced PCA, in that PCA peaked at 6 hr and fell to insignificant levels after 24 hr. When transferred to microtiter plates at this time, they could be restimulated neither with LPS, nor with surface-adherent IgG nor with IFN-γ. Evidence was obtained that the failure to express PCA was due to a refractory state of the cells rather than to the generation of cell-bound or secreted inhibitors of coagulation. The loss of PCA expression could be prevented by pre-exposure to IFN-γ. Thus, PCA expression may be dissociated from other functional and/or activation parameters (e.g. TNF secretion). For the first time, a state in which cells are completely unresponsive to PCA induction has been identified. Should lower LPS concentrations also be found to induce such a refractory state, our results may be of pathophysiological significance.

Spontaneous and lipopolysaccharide-induced expression of procoagulant activity by equine lung macrophages in comparison with blood monocytes and blood neutrophils

The procoagulant activity (PCA) associated with equine bronchoalveolar lavage cells was determined and compared with that expressed by peripheral blood mononuclear cells and neutrophils. Lung cell preparations from horses affected with chronic pulmonary disease were included in all experiments and there was no difference in the qualitative type of response compared with lung cells which were obtained from healthy horses. Significant amounts of PCA were expressed by cells freshly procured from bronchoalveolar lavages of healthy and diseased horses. When adherent lung cells were kept in culture for some time, cell-associated PCA slightly decreased within 4 h, reached its lowest point after approximately 24 h and rose again during the second week of culture. In contrast, freshly isolated blood mononuclear cells or neutrophils expressed little PCA. Following culture for 24 h, mononuclear cells began to express increased PCA levels. Both cultivated lung cells (comprised mainly on alveolar macrophages) and blood mononuclear cells responded to LPS by dramatically increased PCA expression, whereas neutrophils showed a small augmentation of PCA on LPS stimulation. Fresh mononuclear cells and cultivated lung cells differed in their PCA response to LPS in several respects. Blood mononuclear cells were more sensitive to LPS than lung macrophages and responded to a 100-fold lower LPS concentration than the latter. Mononuclear cell-associated PCA peaked 4 h after stimulation whereas that of cultured macrophages continued to increase up to 24 h after stimulation. Lung macrophages cultured in adherence responded to LPS stimulation with a much higher PCA increase than macrophages cultured in suspension, in teflon containers. However, the culture vessel did not influence the PCA expressed by unstimulated cells. PCA expression depended to a large extent on transcription and translation, as evidenced by a 60–85% reduction of PCA in cycloheximide- or actinomycin D-treated, LPS-stimulated lung macrophages. PCA was largely cell-associated; only a small proportion of cell-associated PCA was shed into the medium. The PCA associated with mononuclear cells and with lung macrophages was tissue factor because of its dependence on clotting factor VII and its independence from clotting factor VIII. The expression of PCA by freshly isolated cells, the lower sensitivity to LPS, and the loss of PCA in the first 24 h of cultivation are indicative of in vivo activation of lung macrophages.

Disparate mechanisms of induction of procoagulant activity by live and inactivated bacteria and viruses

This study describes the dose response, time course, and lymphocyte requirements of procoagulant activity (PCA) induction following stimulation of thioglycolate-elicited BALB/c peritoneal macrophages with live and inactivated bacteria (Bacteroides fragilis, Escherichia coli, and Staphylococcus aureus) and murine hepatitis virus type 3 (MHV-3). Induction of PCA by MHV-3 was significantly more rapid and the maximal PCA achieved was significantly greater than by the three bacterial species studied. In relation to induction of PCA by bacteria, the PCA response was more rapid and of greater magnitude with S. aureus and E. coli than with B. fragilis. MHV-3 induced an augmented PCA response at all concentrations of virus studied in a dose-dependent fashion, whereas higher titers of live bacteria (greater than 10(7) CFU/ml) inhibited PCA, suggesting the production of an inhibitory factor. Significant PCA induction was observed when macrophages were incubated with bacteria or virus in the absence of lymphocytes. At low titers of B. fragilis (10(3) CFU/ml), addition of lymphocytes greatly augmented PCA production, whereas at higher titers (10(7) CFU/ml), the addition of lymphocytes only slightly augmented the PCA response. In contrast, MHV-3 induction of PCA was enhanced by the addition of lymphocytes at all concentrations of virus studied, suggesting a lymphocyte-dependent process. Heat-inactivated bacteria were as effective as live bacteria in inducing PCA, suggesting that induction of PCA by bacteria requires only a bacterial surface component. In contrast, UV-inactivated MHV-3 did not induce PCA, suggesting that viral replication is a necessary step in PCA induction. These results suggest that the cellular and metabolic requirements for induction of PCA differ among viral and bacterial pathogens and may partly explain their differences in pathogenicity.

Absence of monocyte procoagulant activity during the immune response to influenza virus.

The ability of mononuclear phagocytes (MPh) to manifest procoagulant activity (PCA) resulting in the formation of fibrin is thought to be a key MPh effector function in tissue repair. The present study addresses the question of whether monocyte PCA is confined to tissue hypersensitivity reactions or is a general correlate of all immune responses. We show here that PCA is not the obligate outcome when the immune system is stimulated. In particular, under in vitro conditions in which a mitogen (phytohemagglutinin) or an antigen (purified protein derivative of tuberculin) elicits good PCA responses, incubation with influenza virus does not result in the generation of PCA, although other parameters of response to the virus appear to be intact. Moreover, influenza virus can cause suppression of PCA when cultures are stimulated with either phytohemagglutinin, purified protein derivative of tuberculin, or endotoxin, conditions which would otherwise result in good PCA responses. This lack of PCA persists throughout the culture period and is not caused by an effect of influenza virus on the viability of either MPh or leukocytes in general.

Lymphoid procoagulant response to bacterial endotoxin in the rat.

A number of species respond to bacterial endotoxin (lipopolysaccharide [LPS]) wherein cells of the monocyte-macrophage lineage are rapidly induced either directly or via T-cell collaboration to initiate the extrinsic coagulation protease pathway. This results in fibrin formation and deposition as well as consumption of plasma coagulation proteins. It has been claimed that this cellular response, basic to the Shwartzman reaction, is lacking in rats and may account for the more limited severity of the Shwartzman reaction in this species. We examined the in vitro lymphoid procoagulant response in Fischer 344, Brown Norway, and Lewis rats. When peripheral blood mononuclear cells (PBM) were stimulated in vitro with LPS, a procoagulant activity (PCA) response was observed when assayed by acceleration of clotting of recalcified human or rat platelet-poor plasma. The response was rapid, with a maximum achieved at 4 h. PCA was not physically dissociated from viable PBM by 5 mM EDTA, which is consistent with the presence of an intrinsic plasma membrane initiator molecule rather than calcium-bound gamma-carboxylated glutamic acid-containing proteases. The induction of monocyte PCA was prevented by incubation of cells with cycloheximide or actinomycin D, implicating a new biosynthetic requirement. Cultivation of PBM with warfarin did not diminish the function of the effector PCA, nor did vitamin K augment the function of the endotoxin-induced PCA, indicating that the functional activity was not attributable to gamma-carboxylated glutamic acid-containing proteins. No inhibition of the cellular PCA molecule was produced by serine protease inhibitors. The LPS-induced PCA appeared to involve a tissue factor-like molecule since both factors X and VII were required in mediating PCA. Isolation of monocytes and T lymphocytes from LPS-stimulated PBM demonstrated that PCA was present in the monocyte-rich fraction. When isolated rat T lymphocytes and monocytes were separately exposed to LPS, PCA was not induced. In contrast, when the cells were combined, LPS induced PCA, indicating that the PCA response involved cellular collaboration between cells present in T lymphocyte and monocyte populations.

Tissue factor induction in human monocytes. Two distinct mechanisms displayed by different alloantigen-responsive T cell clones.

One component of the cellular immune response to antigens is the expression of procoagulant activity (PCA) by monocytes and macrophages. Induction of human monocyte PCA in response to alloantigenic stimulation requires the collaboration of HLA-DR-responsive T cells. In mixed lymphocyte cultures (MLCs), the induction of monocyte tissue factor appears to be mediated exclusively by a T cell-derived lymphokine. We have used a soft agar cloning method to generate alloantigen-responsive T cell clones from MLCs between irradiated Daudi lymphoblastoid cells and human peripheral blood mononuclear cells. Developing clones were screened for the ability to induce PCA in fresh autologous monocytes in response to Daudi stimulator cells. PCA induction was observed with some, but not all, proliferating T cell clones and two modes of induction were apparent. Some T cell clones mediated PCA induction exclusively by lymphokine production, whereas other clones delivered induction signals by direct cellular collaboration with the monocyte effector cells. These two inductive pathways were represented in distinct, non-inclusive functional subsets of T cell clones. Constitutive production of soluble inducer signals was not observed in T inducer clones. The magnitude of the monocyte PCA response increased in response to an increase in the allogeneic stimulator/T clone responder ratio, and third-party allogeneic cells were unable to elicit the PCA-inducing lymphokine signals from T inducer clones. Both modes of induction were shown to generate tissue factor protein activity in monocytes. Collectively, these results suggest that PCA induction can be initiated in response to alloantigens through collaboration with certain OKT3+, OKT4+, OKT8-, OKM1- T inducer clones, and that induction can be mediated by at least two different functional subsets of human T cells. Stimulation with the appropriate alloantigen may elicit both lymphokine and T cell-contact pathways of induction of tissue factor in human monocytes.

Antigen-induced monocyte procoagulant activity. Requirement for antigen presentation and histocompatibility leukocyte antigen-DR molecules.

The present study explores the interactions between lymphocytes and monocytes that are required for expression of procoagulant activity (PCA) by monocytes in response to purified protein derivative of the tubercle bacillus (PPD) or tularemia antigen. The PCA response was antigen specific: peripheral blood mononuclear cells (PBM) from donors sensitive to PPD or tularemia showed an increase in PCA only in response to the sensitizing antigen. The PCA was tissue factorlike in that Factors VII and X were required for expression of the activity, whereas Factor VIII was not. Maximum PCA developed only after 36 to 72 h. Fractionation of PBM into lymphocytes and monocytes after antigenic stimulation demonstrated that greater than 90% of the PCA was associated with monocytes. Isolated monocytes or lymphocytes incubated with sensitizing antigen had the same PCA as control cells. Purified lymphocytes that had been pulsed with antigen were unable to elicit a PCA response from monocytes to which they were added. However, adherent monocytes incubated with antigen, then washed free of unbound protein, were able to trigger lymphocytes to become stimulatory for PCA toward responding monocytes. The development of antigen-specific PCA in PBM could be blocked by including a monoclonal antibody to HLA-DR antigen in the incubation. The antibody had no effect on the clotting assay, on preformed PCA, cell viability, or on stimulatory antigen itself. These results indicate that elaboration of PCA by mononuclear cells may be an intrinsic part of the classical immune response to antigen, and may explain the presence of fibrin in immune lesions, as well as the occurrence of thrombotic complications in many immune disorders.

Dextran as a modulator of immune and coagulation activities in trauma patients

Low M r dextran has been utilized as a prophylactic therapy in treatment of coagulopathy. There is evidence that monocyte dysfunctions are important contributors to hypercoagulability episodes, as well as to immunoincompetence post-trauma. Dextran is a known monocyte modulator. Consequently, we evaluated the efficacy of dextran infusion in moderating immune dysfunction, monocyte aberrations, and hypercoagulability episodes. Twenty-eight trauma patients were randomly divided into two groups. One group of 15 received dextran at 1 g/kg wt/24 hr for 5 days in addition to standard resuscitation and treatment. The control or nontreated patient group received only standard treatment. Trauma patients in the two groups were retrospectively matched by injury severity score (ISS) to ensure comparability. Blood samples were collected daily for some studies and at 3-day intervals for other assays. In vivo coagulation status was evaluated by assessing the changes in intravascular fibrinopeptide A (FPA). Immune reactivity to the mitogen phytohemagglutinin (PHA) was also evaluated. Both monocyte production of plasminogen activator (PA) and monocyte production of procoagulant activity (PCA) have been shown to correspond to and be augmented by monocyte-T lymphocyte interactions. Consequently, monocyte production of plasminogen activator and procoagulant activity were assessed as measures of monocyte immune activity as well as indicators of monocyte function in controlling the balance between fibrinolysis and coagulation. Only patients with ISS of >25 experienced significant immune, coagulation, or monocyte aberrations. Of those having an injury severity score (ISS) score of 25–35, all of the control and two of the dextran patients had significant perturbations in their immune and monocyte functions. There was no significant difference in the ISS score of the nontreated, control group (31.0) versus the dextran group (28.9). The mean monocyte PA response for nontreated patients was 5.6 units while that of the dextran-treated group was 14.2. This is in comparison to a mean ( x ) of 20.6 for normal individuals. The nontreated patient group's x increase in monocyte PCA response (49.2 ± 6.1) was also significantly different from that of the dextran trauma patients (24.5 ± 2.8). All the trauma patients (both dextran treated and nontreated) with ISS >25 showed significantly altered FPA values but the fibrinopeptide A values for the no drug group were more elevated (32.3 ± 4.3) than those of ISS matched trauma patients receiving dextran (15.4 ± 7.2). Low M r dextran administered as a prophylactic treatment may be of some benefit in controlling post-trauma alterations in monocyte-mediated immunoincompetence and monocytemediated hypercoagulability.

A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

Procoagulant activity in renal transplant recipients.

Procoagulant activity (PCA) of leukocytes of renal transplant recipients was studied. This material, which activates coagulation, has previously been shown to be released from macrophages after they interact with mitogen-stimulated or antigen-stimulated T lymphocytes. Under endotoxin-free conditions, PCA of peripheral blood leukocytes, incubated for 90 min in tissue culture, was elevated in postoperative transplant recipients and in many transplant patients tested around the time of a rejection episode. The response to lipopolysaccharide added during culture was also increased in these populations. The PCA response was factor-VII-dependent when tested with washed peripheral blood mononuclear cells (PBMC), but was factor-VII-independent when tested with unwashed PBMC in their original culture medium. The results indicate a possible link between immunologic events and coagulation in transplant recipients.

Regulation of monocyte procoagulant by chemoattractants

Various n-formylated peptides function as receptor-specific chemoattractants for both granulocytes and monocytes. Because these agents are important tools in the study of leukocyte function in vitro, we chose to examine their effects on leukocyte procoagulant activity. The synthetic chemotactic peptide N-formyl-methionyl-leucyl phenylalanine (FMLP) induces a fourfold increase in procoagulant activity (PCA) in cultured human monocytes at an optimal dose of 5 X 10(-9) mol/L, whereas higher doses inhibit PCA response. Although nonadherent lymphocytes are not absolutely required for PCA expression, their presence significantly amplifies monocyte PCA. Irradiation of nonadherent lymphocytes before mixing them with FMLP and adherent cells abolishes their ability to amplify PCA. Kinetic studies demonstrate an increase in optimal dose FMLP-stimulated PCA over time whereas high- dose inhibition of PCA generation occurs at various incubation times. Cell viability is unaffected by inhibitory concentrations of FMLP. Supernates from high-dose FMLP-stimulated cells fail to inhibit later expression of PCA by cells exposed to endotoxin. The cellular procoagulant remains cell-bound and exhibits characteristics of thromboplastin (tissue factor), including inhibition by concanavalin A and phospholipase C as well as the ability to shorten the clotting times of factor VIII but not factor VII-deficient substrate plasmas. These results suggest a complex system of lymphoid cell regulation of procoagulant generation by monocytes exposed to various chemotactic peptides in vitro.

Regulation of Monocyte Procoagulant by Chemoattractants

Various n-formylated peptides function as receptor-specific chemoattractants for both granulocytes and monocytes. Because these agents are important tools in the study of leukocyte function in vitro, we chose to examine their effects on leukocyte procoagulant activity. The synthetic chemotactic peptide N-formyl-methionyl-leucyl phenylalanine (FMLP) induces a fourfold increase in procoagulant activity (PCA) in cultured human monocytes at an optimal dose of 5 X 10(-9) mol/L, whereas higher doses inhibit PCA response. Although nonadherent lymphocytes are not absolutely required for PCA expression, their presence significantly amplifies monocyte PCA. Irradiation of nonadherent lymphocytes before mixing them with FMLP and adherent cells abolishes their ability to amplify PCA. Kinetic studies demonstrate an increase in optimal dose FMLP-stimulated PCA over time whereas high-dose inhibition of PCA generation occurs at various incubation times. Cell viability is unaffected by inhibitory concentrations of FMLP. Supernates from high-dose FMLP-stimulated cells fail to inhibit later expression of PCA by cells exposed to endotoxin. The cellular procoagulant remains cell-bound and exhibits characteristics of thromboplastin (tissue factor), including inhibition by concanavalin A and phospholipase C as well as the ability to shorten the clotting times of factor VIII but not factor VII-deficient substrate plasmas. These results suggest a complex system of lymphoid cell regulation of procoagulant generation by monocytes exposed to various chemotactic peptides in vitro.

The instructor cell for the human procoagulant monocyte response to bacterial lipopolysaccharide is a Leu-3a+ T cell by fluorescence-activated cell sorting.

A variety of responses of cells of the lymphoid system are associated with acquisition of the capacity to initiate the coagulation protease pathways. The initiating or procoagulant molecules are produced by the monocyte; however, a number of studies have indicated that lymphocyte collaboration is required. The induction of human monocyte procoagulant activity (PCA) by the model stimulus bacterial lipopolysaccharide (LPS) was examined in the present study by using relatively highly purified monocyte and lymphocyte populations in reconstitution experiments. Consistent with prior studies, the PCA response could not be generated by highly purified monocytes alone after exposure to LPS. The ability to generate PCA was restored to these monocyte populations by the addition of fibronectin-gelatin nonadherent lymphocytes, nylon wool effluent T cells, or Leu-3a+ inducer/helper T cells selected by fluorescence-activated cell sorting. T cells added to monocytes at a ratio of 8:1 or higher, and Leu-3a+ cells added at a ratio of 6:1 or higher, provided a maximal collaboration for monocyte PCA induction by LPS. These results substantiate further previous suggestions of an absolute requirement for collaborating T cells and demonstrate that these instructor cells carry a marker for the inducer/helper subset.

Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice.

The in vitro induction of procoagulant activity (PCA) in murine peripheral blood mononuclear cells (PBM) by murine hepatitis virus type 3 (MHV-3) correlates with the disease susceptibility in three strains of mice. PBM from BALB/c mice, a strain in which MHV-3 infection results in fatal acute fulminant hepatitis, responds to the virus with a robust PCA response, whereas PBM from C3H/St mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest PCA response. In contrast, PBM from A/J mice, a strain fully resistant to MHV-3, generate no increase in PCA above control levels. The induction phase of MHV-3 PCA is rapid, with an increase within 1-1.5 h, with maximum activity at 18h, and it precedes MHV-3 replication in either 17 CL1 cells, a fully permissive cell line, or in monocytes from these strains of mice. The PCA response of BALB/c PBM exceeds the response to any other known stimulus. No induction occurs upon direct stimulation of monocytes by MHV-3, but in the presence of lymphocyte collaboration, the PCA response is observed first at a lymphocyte:monocyte ratio of 2:1 and reaches a maximum as the lymphocyte:monocyte ratio approaches 4:1. This response appears to provide a functional marker for susceptibility to MHV-3 infection in inbred strains of mice and could be important in the pathogenesis of MHV-3-induced disease.