Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary L-arginine

BACKGROUND: There is evidence that tissue factor (TF) is a major contributor to the thrombogenicity of a ruptured atherosclerotic plaque. Nitric oxide (NO) has antiatherogenic and antithrombotic properties. We investigated whether L-arginine (L-arg), the endogenous precursor of NO, might affect the ability of monocytes to produce TF. METHODS AND RESULTS: We studied TF expression in 18 rabbits with atherosclerosis induced by bilateral iliac damage and 10 weeks of a 2% cholesterol diet. Six weeks after the initiation of the diet, an angioplasty was performed. After angioplasty, the surviving rabbits (n=15) were randomized to receive L-arg (2.25%) supplementation in drinking water (L-arg group, n=8) or no treatment (untreated group, n=7). TF expression was evaluated in mononuclear cells from arterial blood in the presence and absence of endotoxin stimulation. Monocyte TF expression, as assessed with an amidolytic assay, did not differ significantly before or after the induction of atherosclerotic lesions (87+/-15 versus 70+/-12 mU of TF/1000 monocytes, P=NS). Endotoxin-stimulated TF activity increased significantly 4 weeks after angioplasty (138+/-22 versus 70+/-12 mU of TF/1000 monocytes, P=0.02). This increase was blunted by L-arg (43+/-16 mU of TF/1000 monocytes, P=0.01). CONCLUSIONS: This study demonstrates that angioplasty-induced plaque rupture is associated with a marked increase in monocyte TF response that is blunted by the oral administration of L-arg. This suggests that the documented antithrombotic properties of NO may be related in part to an inhibitory effect on monocyte TF response.     

Interleukin-6 and Epstein-Barr virus induction by cyclosporine A: potential role in lymphoproliferative disease

Posttransplant patients undergoing prolonged cyclosporine A (CsA) immunosuppressive therapy have been reported to have increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. We undertook experiments to analyze the possible actions of CsA during EBV-infection of human peripheral blood mononuclear cells (PBMC). EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell outgrowth as compared with those cultured without CsA. PBMC, after infection with EBV and CsA treatment, demonstrated increased interleukin-6 (IL-6) activity in the culture supernatant. The induction of IL-6 appears to differ within the various lymphocyte populations. In monocytes, IL-6 expression appears preferentially induced by EBV and is initiated by the binding of the two major virion glycoproteins, gp350 and gp220. Expression of IL-6 in T cells appears to be due mainly to CsA. B cells also express IL-6 after EBV exposure, but not after CsA treatment. EBV-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which is induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. CsA, in promoting both increased numbers of lytic EBV B cells and an EBV paracrine factor, IL-6, within the microenvironment of EBV B cell:T cell and EBV B cell:monocyte interactions, may result in increased EBV B-cell immortalization and ultimately lead to the promotion of B-cell lymphomas in immunosuppressed patients.     

Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production

BACKGROUND: Cyclosporine (CSA) has improved patients and organ-graft survival rates, but its chronic nephrotoxicity is still an issue. Although prolonged vasoconstriction could contribute to chronic CsA tubulointerstitial changes by producing chronic ischemia, this relationship has been difficult to demonstrate thus far, and cellular origin and mediators of these structural alterations remain ill-defined. METHODS: As a part of a clinical trial in kidney transplant recipients on triple immunosuppressive therapy (CsA, azathioprine and steroid), which includes renal biopsy as "per protocol," 22 patients enrolled between 12 and 24 months posttransplantation underwent renal hemodynamic evaluation by measuring glomerular filtration rate and renal plasma flow by the plasma clearance of unlabeled iohexol and the renal clearance of para-aminohippuric acid, respectively. In parallel, the CsA pharmacokinetic profile was also determined. A week later, a protocol biopsy of kidney graft was performed. Light microscopy examination and localization of endothelin-1, RANTES, monocyte chemoattractant protein-1 gene expression by in situ hybridization in the graft specimens were evaluated and related to the pattern of histologic lesions. RESULTS: Ten out of 22 kidney transplant recipients who underwent the protocol biopsy had CsA nephrotoxicity, eight had chronic rejection, and four had no lesions at histological examination. The total daily exposure to CsA was higher in patients with CsA nephrotoxicity than in those with chronic rejection or no lesions at biopsy. Renal function was preserved in the CsA toxicity group as compared with the chronic rejection group, despite some degree of renal hypoperfusion. Tubular atrophy and striped interstitial fibrosis were found in all patients with light microscopical evidence of CsA nephrotoxicity, whereas glomerular and arteriolar lesions were less frequent. Intense staining for endothelin-1, RANTES, and monocyte chemoattractant protein-1 mRNAs selectively localized at tubular epithelial cells was found in biopsies taken from patients with CsA nephrotoxicity, but not in the chronic graft rejection group, whose tubuli had only minimal staining for RANTES mRNA on a few occasions. CONCLUSION: Long-term CsA administration to kidney allograft recipients leads to tubulointerstitial injury independently of its vascular effect. The possible contribution to the development of interstitial fibrosis of inflammatory and growth factors released by tubular cells in which CsA accumulates is proposed.     

Enhancement of monocyte procoagulant activity by adhesion on vascular smooth muscle cells and intercellular adhesion molecule-1-transfected Chinese hamster ovary cells

BACKGROUND: Plaque erosion is a frequent finding in sudden death due to coronary thrombosis. The present study sought to investigate whether monocyte adhesion to human aortic vascular smooth muscle cells (VSMCs) induces procoagulant activity (PCA) and whether this could be mediated by intercellular adhesion molecule-1 (ICAM-1). METHODS AND RESULTS: We incubated mononuclear cells (MNCs) with VSMCs and ICAM-1-transfected Chinese hamster ovary (CHO) cells, investigated monocyte tissue factor (TF) mRNA expression by Northern blot analysis and TF protein expression by ELISA, and measured PCA. Incubation of MNCs with VSMCs for 6 hours increased PCA from 0.7+/-0.1 to 166.0+/-37.9 mU/105 cells (P=0.007), which could be inhibited in a dose-dependent manner by the addition of blocking anti-ICAM-1 monoclonal antibodies. Prestimulation of VSMCs with interleukin-1beta enhanced surface ICAM-1 expression significantly but did not induce PCA in VSMCs. Incubation of MNCs with prestimulated VSMCs led to a further increase in PCA to 239.9+/-27.9 mU/10(5) cells (P=0.02 compared with incubation with unstimulated VSMCs). Incubation of MNCs with VSMCs enhanced TF mRNA after 2 hours and significantly increased TF protein content after 6 hours. Incubation of purified monocytes with ICAM-1-transfected CHO cells increased PCA from 1.2+/-0.2 to 81.9+/-3.3 mU/10(5) cells (P<0.001 compared with incubation with untransfected CHO cells) after 6 hours. This effect could be inhibited significantly by the addition of blocking anti-CD18, anti-CD11b, or anti-CD11c monoclonal antibodies. Similar results were obtained for MNCs. CONCLUSIONS: Monocyte adhesion to VSMCs induces TF mRNA and protein expression and monocyte PCA, which is regulated by beta2-integrin-mediated monocyte adhesion to ICAM-1 on VSMCs.     

Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis through nuclear factor-kappa B activation and monocyte chemoattractant protein-1 synthesis

Angiotensin-converting enzyme (ACE) inhibitors reduce macrophage infiltration in several models of renal injury. We approached the hypothesis that angiotensin II (AngII) could be involved in inflammatory cell recruitment during renal damage through the synthesis of monocyte chemoattractant protein-1 (MCP-1). In a model of immune complex nephritis, we observed an up-regulation of renal MCP-1 (mRNA and protein) coincidentally with mononuclear cell infiltration that were markedly reduced by treatment with the ACE inhibitor quinapril. Exposure of cultured rat mesangial cells to AngII increased MCP-1 mRNA expression (2.7-fold) and synthesis (3-fold), similar to that observed with TNF-alpha. Since NF-kappaB is involved in the regulation of MCP-1 gene, we explored whether the effects of AngII were mediated through NF-kappaB activation. Untreated nephritic rats showed increased renal NF-kappaB activity (3.5-fold) that decreased in response to ACE inhibition. In mesangial cells, AngII activated NF-kappaB (4.3-fold), and the NF-kappaB inhibitor pyrrolidine dithiocarbamate abolished the AngII-induced NF-kappaB activation and MCP-1 gene expression. Our results suggest that AngII could participate in the recruitment of mononuclear cells through NF-kappaB activation and MCP-1 expression by renal cells. This could be a novel mechanism that might further explain the beneficial effects of ACE inhibitors in progressive renal diseases.     

Donor cell induced CD69 expression and intracellular IL-2 and IL-4 production by peripheral blood lymphocytes isolated from kidney transplant recipients

Flow cytometry assays, which measure CD69 activation and intracellular cytokine production, have been used to measure peripheral blood lymphocyte (PBL) responses to in vitro antigen exposure. In the present study, we show that, in healthy individuals and immunosuppressed kidney transplant recipients, CD69 expression and intracellular cytokine production by peripheral blood T cells compare favorably to thymidine uptake as a measure of PBL response to alloantigen in mixed leukocyte culture (MLC). Heparinized whole blood from 23 healthy individuals was incubated for 24-48 h with 3rd party allogeneic monocytes; blood from twelve kidney transplant recipients was incubated with monocytes from their kidney donor and with monocytes from unrelated individuals. The percentage of T cells expressing surface CD69 or intracellular IL-2 or IL-4 was determined by 3-color flow cytometry. We identified 5 donor-specific response patterns in our kidney transplant group. One transplant recipient was hyporesponsive; his cells did not express CD69 or produce IL-2 in response to either donor or 3rd party allogeneic cells. All other transplant recipients expressed CD69 and IL-2 in response to 3rd party allogeneic cells. Two had no response to donor cells (donor-specific hyporesponsiveness), three had donor-specific anergy (CD69 expression without cytokine production in response to donor cells), five had a donor-specific Thl response (CD69 expression and IL-2 production in response to donor cells), and one had a donor-specific Th2 response (CD69 expression and IL-4 but not IL-2 production in response to donor cells). Rapid measures of donor-specific hyporesponsiveness such as CD69 activation antigen expression and intracellular cytokine production may prove valuable in monitoring lymphocyte function and aid in the long-term management of kidney transplant recipients.     

Prevention of graft-versus-host disease and the induction of transplant tolerance by low-dose UV-B irradiation of BM cells combined with cyclosporine immunosuppression

GVHD is prevented and stable chimerism is induced in the rat BMT model by 700 J/m2 but not 100-500 J/m2 UV-B irradiation of allogeneic BM cells. Paradoxically, CsA which prevents GVHD in clinical BMT causes an aggressive autoimmune disease termed syngeneic GVHD in irradiated syngeneic BMT recipients after its withdrawal. Recently, we have shown that while 500-700 J/m2 UV-B irradiation of syngeneic BM cells combined with a 30-day course of CsA recipient immunosuppression impairs hemopoiesis due to lack of hemopoietic factors, a low dose of 100-300 J/m2 UV-B is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment. This study extends these findings to the P-to-F1 hybrid and fully allogeneic rat BMT models and examines the effectiveness of low-dose UV-B irradiation of BM cells combined with a short course of CsA treatment in the prevention of GVHD and induction of transplant tolerance. Lethally gamma-irradiated (10.5 Gy) LBNF1 recipients of naive or UV-B irradiated (100-700 J/m2) BMT were treated with CsA (12.5 mg/kg/day) for 30 consecutive days after BMT. All lethally irradiated LBNF1 that did not receive BMT died in < 16 days, while animals transplanted with UV-B (700 J/m2) BMT survived > 1 year without GVHD. In contrast, all recipients of naive BMT died of lethal GVHD in < 50 days. Similarly, all recipients of naive BMT that received a 30-day course of CsA therapy developed severe GVHD with 60% mortality after cessation of CsA therapy. CsA-treated recipients of BMT irradiated with 700 J/m2 died between 12 and 25 days from failure of hemopoiesis. In contrast, CsA-treated recipients of 100-200 J/m2 UV-B irradiated BMT showed full BM engraftment without GVHD after cessation of CsA and survived > 1 year. These results were reproducible in the fully allogeneic UV-B BMT model. To test for donor-specific tolerance, the animals challenged 100 days after BMT with cardiac allografts accepted permanently (> 100 days) Lewis but not BN (non-BMT parental donor) cardiac allografts. Our results confirm that 700 J/m2 UV-B irradiation of BM cells combined with CsA recipient immunosuppression impairs the recovery capacity of stem cells while the use of lower UV-B (100-200 J/m2) is effective in preventing CsA-induced autoimmune disease without endangering BM engraftment and leads to induction of transplant tolerance.     

Modulation of [3H]dopamine release by neuropeptide Y in rat striatal slices

Polycythemia vera (PV) is associated with a high incidence of thrombosis. The association of apparent and secondary polycythemia with thrombosis is not clear. It was suggested that activation of the coagulation system contributes to thrombus formation in PV. However, the mechanism of activation is unknown. Monocytes generate a potent tissue factor (TF) upon stimulation with various substances, which is involved in thrombus formation in various disorders. Therefore, we studied the possibility that the factor is involved in the activation of coagulation and thrombus formation also in PV. Unstimulated peripheral blood mononuclear cells (PBMC) from each of the different types of polycythemia expressed weak TF activity (2 U) and antigen (41.4 to 52.9 pg/ml), which were similar to normal controls. Following stimulation with endotoxin, PBMC from normal controls and from apparent and secondary polycythemia showed a 3.9- to 4.5-fold increase in TF, while cells from PV showed a 21-fold increase (P<0.001). Similar levels were generated by PBMC after treatment of PV and at the spent phase. TF was generated by monocytes but not by lymphocytes. Plasma prothrombin fragment1+2 (F1+2) levels, assayed at the same time, were significantly higher in PV (2.46 nm) compared to normals and apparent and secondary polycythemia (0.22 to 0.32 nm), and were in a significant correlation with monocyte TF activity and antigen levels (r = 0.77, 0.87). The high levels of F1+2 confirm that the coagulation system is activated in PV. The increased capacity of monocytes to generate TF may be responsible for the activation of the coagulation system and thrombus formation. The hypercoagulability state that is induced by this mechanism suggests that long-life oral anticoagulation should be considered once thrombosis has been developed in PV.     

Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro

An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.     

Twenty-four-hour profiles of luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol levels: a semilongitudinal study throughout puberty in healthy boys

Monocytes and endothelial cells interact at sites of vascular injury during inflammatory response, thrombosis, and development of atherosclerotic lesions. Such interactions result in modulation of several biological functions of the two cell types. Because both cells, on appropriate stimulation, synthesize tissue factor (TF), we examined the effect of human umbilical vein endothelial cell (HUVEC)/monocyte coculture on the expression of TF. We found that the coincubation resulted in TF generation, which was maximal at 4 hours, increased with increasing numbers of monocytes, and required mRNA and protein synthesis. Supernatant from HUVEC/monocyte coculture induced TF activity in HUVECs, but not in monocytes, indicating that HUVEC were the cells responsible for the activity, and that soluble mediators were involved. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), well-known inducers of TF in HUVECs, were found in the supernatant from the coculture, and specific antibodies directed against either cytokine inhibited TF generation. The need of IL-1 beta and TNF-alpha synthesis in order to elicit TF expression was also suggested by the delay observed in TF mRNA formation and TF activity generation when monocytes were incubated with HUVECs. IL-1 beta and TNF-alpha antigen levels in the coculture supernatant, and, consequently, HUVEC TF expression, were inhibited in the presence of anti-CD18 monoclonal antibody. These findings emphasize the role of cell-cell contact and cross-talk in the procoagulant activity, which could be responsible for the thromboembolic complications observed in those vascular disorders in which monocyte infiltration is a common feature.     

In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mumol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mumol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.     

IL-1beta-induced monocyte chemoattractant protein-1 gene expression in endothelial cells is blocked by proteasome inhibitors

Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1beta induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1beta induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1beta-induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-kappaB-mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-kappaB translocation to the nucleus, norLeu had no effect on NF-kappaB nuclear translocation or IL-1beta-induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1beta-induced MCP-1 gene expression in human endothelial cells.     

Leukocyte phenotypic changes in an in vitro model of ABO hemolytic transfusion reaction

BACKGROUND: ABO antigen-antibody interaction in the presence of peripheral blood leukocytes (white cells) results in the production of a variety of proinflammatory cytokines. However, although tumor necrosis factor alpha has been shown to be derived at least primarily from monocytes, the range of cells activated by this process has not previously been reported. Therefore, changes in mononuclear cell surface antigen expression were studied, to determine which subsets of white cells appeared to be activated in the setting of ABO incompatibility. STUDY DESIGN AND METHODS: Group O peripheral blood mononuclear cells (PBMCs) were incubated in autologous plasma with group A or O red cells (RBCs) for up to 24 hours. White cell expression of activation and adhesion markers was measured at 2 and 24 hours by flow cytometry, using direct or indirect fluorescein or phycoerythrin labeling. RESULTS: Expression of lymphocyte activation markers CD25, CDw108, and CD109 was equivalent when PBMCs incubated with group A and O RBCs were compared. However, after 2 hours, mean fluorescence of CD14 on PBMCs incubated with group A RBCs was 65 percent of that on PBMCs incubated with group O RBCs and remained similarly decreased at 24 hours. CD44 expression was upregulated on PBMCs exposed to both group A and O RBCs, but it was increased significantly more on monocytes exposed to group A RBCs. The ability to bind hyaluronic acid was induced in approximately 42 percent of CD14+ monocytes exposed to group A RBCs but in no cells exposed to group O RBCs. CONCLUSION: Downregulation of CD14 and increased binding of hyaluronic acid reflects monocyte activation in this model. No evidence of lymphocyte activation was found, supporting the hypothesis that ABO transfusion reactions primarily activate monocytes.     

Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.     

A 16-element phased-array head coil

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.     

Control of monocyte influx in glomerulonephritis in transplanted kidneys in the rat

Induction of anti-Thy-1 nephritis in different strains of inbred rats results in phenotypically different types of renal diseases. In Wistar and Lewis (LEW) rats, a transient influx of ED1+ macrophages occurs 24 hours after injection of anti-Thy-1 antibodies, whereas this does not occur in F344 rats. The present experiments were designed to investigate the role of the kidney in the regulation of the monocyte influx in this model. To dissociate the role of the immune system from local intrarenal factors in the control of monocyte influx, anti-Thy-1 nephritis was induced in LEW rats with an F344 kidney transplant and in F344 rats with a LEW kidney allograft. Acute rejection episodes were prevented by treatment with an anti-CD4 monoclonal antibody. Control rats received a syngeneic kidney graft. Monocyte influx after injection of anti-Thy-1 antibodies was found in the glomeruli of both LEW and F344 kidneys removed from LEW recipients, whereas there was no demonstrable monocyte influx after infusion of anti-Thy-1 antibodies in either LEW or F344 kidneys removed from F344 recipients. Monocyte infiltration correlated with the subsequent expansion of the mesangial extracellular matrix. The inability to attract monocytes was not due to the lack of glomerular expression of chemokines, because F344 and LEW glomeruli demonstrated a similar expression of monocyte chemoattractant protein-1 (MCP-1). Differences in the ability to activate the complement system were excluded. We conclude that the immune system controls the glomerular influx of monocytes and that the reaction of the mesangial cells is probably controlled by combinations of cytokines produced during the inflammatory process.     

Effects of ischaemia-reperfusion and cyclosporin-A on cardiac muscle ultrastructure

The present study investigates the effects on the cardiac muscle cell of two of the determining factors for the success of organ transplant; ischaemia-perfusion and immunosuppressive treatment with cyclosporin-A (CsA). To this end an abdominal, heterotopic heart transplant model in singenic Sprague-Dawley rats was employed. Three study groups were established: Group I (control, n = 15) animals undergoing heart transplant without treatment; Group II (n = 15) animals undergoing heart transplant and subjected to a daily dose of CsA in a cremophor vehicle (Sandimun) (5 mg/kg/sc); Group III (n = 15): animals undergoing heart transplant and administered a daily dose of pure CsA (5 mg/kg/sc). Recipient animals were sacrificed 7, 14, 21, 30 and 50 days after transplant. During the post-operative period, heart function was assessed by daily abdominal palpation. Graft specimens obtained at each follow-up period were subjected to light and transmission electron microscopy. Immunohistochemical analysis of specimens was performed using the rat macrophage-specific monoclonal antibody MCA-341. The ischaemia/reperfusion process induced considerable alteration to cardiac muscle cells of control animals. Effects, apparent after the first week of transplant, included mitochondrial swelling and loss of cristae, hypertrophy of the sarcoplasmic reticulum and structural changes to sarcomeres. Two weeks after transplant, the myocardium was infiltrated by inflammatory cells. These effects diminished 30 days post-transplant. Cardiac tissues of treated animals (groups II and III) showed similar behaviour although, in the latter group, mitochondrial damage was greater and intense myocardial fibrosis took place. Infiltration of cardiac muscle by white blood cells did not take place until 3 weeks post-implant. These results indicate: a) The ultrastructural changes detected in cardiac fibres of animals of the three study groups were attributable to the ischaemia/reperfusion process rather than to treatment with CsA; b) CsA appears to augment mitochondrial damage and myocardial fibrosis; c) the inflammatory response was delayed and reduced by the immunosupressant; and d) the cremophor administration vehicle did not seem to exert an independent toxic effect on the myocardium.     

Quantitation of immunosuppression by flow cytometric measurement of the capacity of T cells for interleukin-2 production

BACKGROUND: Methods to quantitate the effects of immunosuppressive drugs on immune reactivity might be helpful for monitoring immunosuppressive treatment. Cyclosporine (CsA) inhibits the induction of cytokine synthesis in T cells, and measurement of interleukin (IL)-2 production might constitute a parameter of this drug's effect. METHODS: We determined the percentages of CD4+ and CD8+ lymphocytes producing IL-2 upon stimulation by phorbol myristate acetate and calcium ionophore in whole blood culture, using immunostaining of intracytoplasmatic and membrane markers, followed by multiparameter flow cytometry. A total of 38 clinically stable transplant patients on various immunosuppressive protocols were studied. RESULTS: The percentage of CD4+ T cells producing IL-2 was strongly reduced in patients compared with healthy controls (23% [range, 3-68%] vs. 59.0% [range, 41-70%]; P=0.000035). The percentage of CD4+ T cells producing IL-2 was negatively correlated with the CsA level (Rc=-0.0821, P=0.00002297) but not with prednisolone or azathioprine doses. Fewer CD8+ T cells produced IL-2 in transplant patients compared with controls, but the difference failed to reach statistical significance. The percentage of CD8+ T cells capable of producing IL-2 was inversely correlated to CsA levels (Rc=-0.0375, P=0.0011). CONCLUSIONS: These data suggest that the functional effects of CsA in transplant recipients can be quantitatively determined and that the capacity of CD4+ T cells to produce IL-2 upon stimulation constitutes a functional parameter of CsA effects on the immune system. Prospective studies are required to determine whether this method is useful for clinical monitoring.