Abnormal haemostasis and blood viscosity in malignant hypertension.

We have previously shown abnormalities of haemostasis suggestive of intravascular coagulation in patients with malignant hypertension, a condition associated with retinopathy and renal fibrin deposition. To determine whether such abnormalities are specific to malignant hypertension, we have measured several haemostatic and haemorheological variables in 18 patients with malignant hypertension (Group 1), 18 matched healthy controls (Group 2), and 18 patients with non-malignant hypertension (Group 3) matched for renal pathology, blood pressure and serum creatinine with Group 1. Both Groups 1 and 3 had increased mean levels of fibrinogen, factor VIIIc, beta-thromboglobulin, plasma viscosity and blood viscosity (corrected for haematocrit); and decreased mean levels of haematocrit, antithrombin III and platelet count. Mean levels of fast antiplasmin and alpha2-macroglobulin were elevated in Group 1 but not in Group 3. We conclude that most blood abnormalities are not specific to malignant hypertension; are also present in patients with non-malignant hypertension who have similar levels of blood pressure and renal damage; and might result from renal damage as well as promoting further renal damage by enhancing fibrin deposition. However increased levels of fibrinolytic inhibitors in malignant hypertension merit further investigation in relation to removal of renal fibrin.

Stimulation of inflammatory responses in vitro and in vivo by lipophilic HMG-CoA reductase inhibitors.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalyses the rate limiting step in cholesterol biosynthesis and is markedly inhibited by the statin family of drugs. The effect of statins on lipid lowering is clearly defined, but the ability of the drugs to directly regulate inflammatory functions has not been well explored. In this report, we show that there are differences among the statins in their capacity to induce proinflammatory responses both in human monocytes in vitro, and in leukocytes in mice in vivo. Treatment of human monocytes with lipophilic statins alone stimulated the production of MCP-1, IL-8, TNF-alpha and IL-1 beta and markedly sensitized the cells to subsequent challenge with inflammatory agents. Lipophilic statins also increased the production of reactive oxygen species in monocytes. In contrast, pretreatment of cells with the hydrophilic pravastatin did not induce these heightened inflammatory responses. Furthermore, treatment of mice with lipophilic statins caused a markedly higher influx of leukocytes into the inflamed peritoneal cavity following challenge with thioglycollate. Overall, these results demonstrate that the lipophilic statins influence a regulatory pathway in monocytes that controls cytokine production and that the statins induce different pro-inflammatory responses both in vitro and in vivo.

Physical associations between CD45 and CD4 or CD8 occur as late activation events in antigen receptor-stimulated human T cells.

Activation of human PBL T cells with solid phase anti-CD3 mAb or during the course of an MLR response gives rise to the association of CD4 or CD8 molecules with the protein tyrosine phosphatase, CD45, on the cell surface. This paired association of cell-surface molecules occurs late in the activation cycle and appears to be dependent upon Ti-CD3-mediated signaling because mitogen-driven activation does not induce formation of the complex. Maximal association occurred 72 to 96 h after exposure to anti-CD3 mAb on both CD4+ and CD8+ T cells. In contrast, association between CD8 and CD45 during an MLR response did not occur until day 6 of a MLR whereas CD4-CD45 association was detected by 72 h of culture. The kinetics of association between CD4 or CD8 and CD45 was measured by fluorescence resonance energy transfer and confirmed by immunoprecipitation of dithiobis succinimidylpropionate or disuccinimidyl suberate cross-linked 125I-labeled resting or activated T cells. The molecules that co-precipitated with either CD4 or CD8 and had an apparent kDa of 180 to 205 could be immunodepleted with anti-CD45 mAb. Furthermore, CD4 or CD8 immunoprecipitates from 96-h activated T cells contained significant levels of protein tyrosine phosphatase activity whereas corresponding immunoprecipitates from resting or recently activated T cells showed little protein tyrosine phosphatase activity. This association may allow CD45 to engage and dephosphorylate lck or another CD4- or CD8-associated substrate in order to reset the receptor complex to receive a new set of stimuli. Our observations suggest that synergistic signaling provided as a consequence of CD4 or CD8 association with the TCR after antigenic stimulation may develop on a different temporal scale than that observed after soluble anti-CD4+ anti-CD3 heteroconjugate antibody cross-linking.

Stimulation of tyrosine phosphorylation and calcium mobilization by Fc gamma receptor cross-linking. Regulation by the phosphotyrosine phosphatase CD45.

The binding and subsequent cross-linking of murine IgG2a or human IgG to the Fc gamma R on the monocytic cell line THP-1 induced a rapid, dose-dependent increase in tyrosine phosphorylation of several proteins (a doublet centered around 110 kDa, and bands at 80, 60, and 52 kDa) and smaller increases in other proteins. This phosphorylation was accompanied by an increase in intracellular free Ca2+. The signaling required the cross-linking of the IgG, either through a biotin-avidin complex or with a F(ab')2 second antibody. Cross-linking of an F(ab')2 fragment of mAb 32.2 to Fc gamma RI (CD64) or an Fab fragment of mAb IV.3 to Fc gamma RII (CDw32) gave similar results to those observed with intact murine IgG2a or human IgG. Cross-linking of a F(ab')2 fragment of mAb 3G8 to Fc gamma RIII (CD16) had very little effect. Increases in both tyrosine phosphorylation and intracellular free Ca2+ were significantly reduced in a dose-dependent manner upon treatment of THP-1 cells with the tyrosine kinase inhibitors herbimycin-A, genistein, or erbstatin. Additionally, there was a marked inhibition of both Ca2+ mobilization and tyrosine phosphorylation when a F(ab')2 fragment of a mAb (T191) to the protein tyrosine phosphatase CD45, was co-cross-linked with either Hu-IgG, Mu-IgG2a, F(ab')2 anti-Fc gamma RI, or Fab anti-Fc gamma RII. Taken together these results suggest that signaling through Fc gamma RI (CD64) and Fc gamma RII (CDw32) in the monocytic leukemia cell line THP-1 gives rise to rapid tyrosine phosphorylation of several proteins followed by an increase in intracellular calcium. In addition, CD45 is able to inhibit the intracellular signaling when it is brought into close proximity to the Fc gamma R. This suggests that this transmembrane tyrosine phosphatase may regulate the stimulation of the cells through the Fc gamma R.

Effect of interferon-gamma on antigen processing in human monocytes.

The effect of interferon-gamma (IFN-gamma) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fc gamma receptor (Fc gamma R) was followed for various times after treatment of cells with IFN-gamma. THP-1 cells that had been treated with IFN-gamma for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-gamma treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48-72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-gamma, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-gamma-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-gamma modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.

Cross-linking of Fc gamma receptor to surface immunoglobulin on B cells provides an inhibitory signal that closes the plasma membrane calcium channel.

Stimulation of B lymphocytes by the cross-linking of surface Ig (sIg) with an F(ab')2 antibody fragment leads to the rapid activation of several tyrosine kinases. This gives rise to the activation of phospholipase C gamma (PLC gamma) and the generation of inositol phosphates. These, in turn, lead to a prolonged elevation of intracellular Ca2+ ([Ca2+]i) consisting of a rapid release of Ca2+ from intracellular stores and a sustained influx of extracellular Ca2+. In contrast, co-cross-linking sIg to Fc gamma receptor (Fc gamma RII) with intact anti-sIg induces a much more transient increase in [Ca2+]i. Stimulation of the murine B cell lymphoma, A20, with F(ab')2 anti-sIgG leads to the production of high levels of IL-2, while co-cross-linking of sIgG with Fc gamma RII blocks this response. In studies reported here, we show that co-cross-linking of Fc gamma RII with sIg prevents the influx of extracellular Ca2+ without significantly affecting the tyrosine phosphorylation of substrates including PLC gamma 1, PLC gamma 2, and Syk or the mobilization of Ca2+ from intracellular stores. In cells that had been previously activated with F(ab')2 anti-IgG, co-cross-linking of sIg to Fc gamma RII rapidly abrogated the influx of extracellular Ca2+ by closing the plasma membrane Ca2+ channel. Additionally, even 2-3 h after stimulation of the cells with F(ab')2 fragment, addition of intact anti-IgG to the cells, or removal of extracellular Ca2+, markedly inhibited (> 90%) IL-2 production. These results indicate that co-cross-linking sIg with Fc gamma RII both prevented the opening of and actively closed the Ca2+ channel, and, through this mechanism, Fc gamma RII was able to control production of IL-2. Overall, since influx of extracellular Ca2+ has been found to be necessary for the proliferation and differentiation of B cells, Fc gamma RII may play a critical role in controlling these responses by regulating the opening of the Ca2+ channel.

Immune complexes of LDL induce atherogenic responses in human monocytic cells.

The ability of immune complexes of LDL or acetylated LDL (acLDL), together with antibodies to LDL, to induce a proatherogenic phenotype in human monocytic cells has been explored. Treatment of THP-1 monocytic cells or peripheral human monocytes with LDL immune complexes containing intact anti-LDL markedly enhanced the ability of these cells to subsequently bind and take up LDL, whereas aggregated LDL or LDL immune complexes prepared with F(ab')2 fragments of anti-LDL had no significant effect. Activation of THP-1 cells with intact LDL immune complexes also stimulated mRNA expression for the scavenger receptor. Additionally, activation of THP-1 cells with insoluble immune complexes of LDL or LDL stimulated generation of reactive oxygen intermediates that, in turn, could oxidize exogenous LDL. These results indicate that the binding of lipoprotein immune complexes to Fc receptors on monocytic cells activates a series of responses that could accelerate the initiation or progression of atherosclerosis.

Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes.

CD40 is a glycoprotein of about 50 kDa that plays a crucial role in B cell growth and differentiation. It is found on the surface of B cells, follicular dendritic cells, monocytes, and some endothelial, epithelial, and carcinoma cells. Engagement of CD40 with anti-CD40 mAbs, gp39 expressed on the cell surface or soluble forms of gp39, primes B cells to efficiently respond to subsequent stimulatory signals leading to B cell proliferation, differentiation, and isotype switching. Peripheral monocytes also express CD40 on the cell surface and expression in increased following treatment with IFN-gamma. Using a soluble murine CD8/human gp39 fusion protein (sgp39) we have found that CD40 plays a crucial role in the regulation of monocyte function. Stimulation of human peripheral monocytes with sgp39 induced homotypic aggregation and significantly increased the expression of several cell-surface proteins including CD54, MHC class II, CD86, and CD40. Soluble gp39 also dramatically enhanced monocyte survival, preventing the onset of apoptosis that normally occurs upon withdrawal of serum. Finally, in the absence of any costimulatory molecules, sgp39 stimulated monocytes to produce TNF-alpha, IL-1 beta, IL-6, and IL-8. These results suggest that ligation of CD40 on human monocytes induces phenotypic changes that would be expected to influence T cell activation by the monocyte and also to enhance or prolong inflammatory responses.

Cross-linking of Fc gamma receptor I (Fc gamma RI) and receptor II (Fc gamma RII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase.

Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (Fc gamma RI) or Fc gamma receptor II (Fc gamma RII) gave rise to the rapid phosphorylation of multiple intracellular proteins. The pattern of proteins that were phosphorylated appeared to be identical. Analysis of these proteins by specific immunoprecipitation indicated that stimulation through either receptor did indeed give rise to phosphorylation of the same set of proteins. These included: Fc gamma RII, phospholipase C (PLC) gamma 1, PLC gamma 2, Vav, GAP, and a protein that co-precipitated with the Fc gamma receptors and migrated with a molecular weight of about 70,000. Co-cross-linking an F(ab')2 anti-CD45 monoclonal antibody together with monoclonal antibodies to either of the Fc gamma receptors inhibited phosphorylation of all these proteins. Analysis of the tyrosine kinases in the cells revealed that both receptors stimulated the phosphorylation and activation of a kinase recognized by antibodies to Syk. Furthermore, the Syk kinase became associated with the Fc gamma RII following receptor cross-linking. These data indicate that although the two Fc gamma receptors have different cytoplasmic tails, they are coupled to the same signal transduction cascade that is regulated by CD45 and involves the activation of Syk.

Human monocytic cells contain high levels of intracellular Fas ligand: rapid release following cellular activation.

Human monocytes express both Fas and Fas ligand (FasL) on the cell surface, and the interaction of these molecules induces spontaneous apoptosis. In this report we present a study of monocytic cells by FACS and confocal microscopy using anti-FasL Abs that reveals high levels of preformed FasL within the intracellular compartment. Further analysis by immunoblotting of cell cytoplasmic proteins confirmed the presence of a 37-kDa protein recognized by anti-FasL Abs. Stimulation of the monocytic cells with immune complexes, PHA, or superantigen gave rise to the rapid release of soluble FasL from within the cells. The presence of high levels of FasL within human monocytes suggests that, upon stimulation, the cells can rapidly translocate intracellular FasL to the cell surface and release it into the extracellular milieu. These findings indicate a novel mechanism for monocytes to respond rapidly to environmental changes, resulting in the release of active, soluble FasL.

A D-amino acid peptide inhibitor of NF-kappa B nuclear localization is efficacious in models of inflammatory disease.

The transcription factor NF-kappa B regulates many genes involved in proinflammatory and immune responses. The transport of NF-kappa B into the nucleus is essential for its biologic activity. We describe a novel, potent, and selective NF-kappa B inhibitor composed of a cell-permeable peptide carrying two nuclear localization sequences (NLS). This peptide blocks NF-kappa B nuclear localization, resulting in inhibition of cell surface protein expression, cytokine production, and T cell proliferation. The peptide is efficacious in vivo in a mouse septic shock model as well as a mouse model of inflammatory bowel disease, demonstrating that NF-kappa B nuclear import plays a role in these acute inflammatory disease models.