Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice.

Hyperhomocysteinemia is a risk factor for stroke, myocardial infarction, and venous thrombosis. Moderate hyperhomocysteinemia is associated with impaired endothelial function, but the mechanisms responsible for endothelial dysfunction in hyperhomocysteinemia are poorly understood. We have used genetic and dietary approaches to produce hyperhomocysteinemia in mice. Heterozygous cystathionine beta-synthase-deficient mice (CBS +/-), which have a selective defect in homocysteine transsulfuration, and wild-type (CBS +/+) littermates were fed either a control diet or a diet that is relatively deficient in folic acid for 6 wk. Plasma total homocysteine was 5.3 +/- 0.7 microM in CBS +/+ mice and 6.4 +/- 0.6 microM in CBS +/- mice (P = 0.3) given the control diet. Plasma total homocysteine was 11.6 +/- 4.5 microM in CBS +/+ mice and 25.1 +/- 3.2 microM in CBS +/- mice (P = 0.004) given a low-folate diet. In mice fed the control diet, relaxation of aortic rings in response to the endothelium-dependent vasodilator acetylcholine did not differ significantly between CBS +/+ mice and CBS +/- mice. In contrast, in mice fed a low-folate diet, maximal relaxation to acetylcholine was markedly impaired in CBS +/- mice (58 +/- 9%) compared with CBS +/+ mice (84 +/- 4%) (P = 0.01). No differences in relaxation to the endothelium-independent vasodilator sodium nitroprusside were observed among the four groups of mice. These data indicate that CBS-deficient mice are predisposed to hyperhomocysteinemia during dietary folate deficiency, and moderate hyperhomocysteinemia is associated with marked impairment of endothelial function in mice.

Eosinophils isolated by magnetic cell sorting respond poorly to lipid chemoattractants.

BACKGROUND: Eosinophils play an important role in allergic inflammation. In vitro methods to isolate human eosinophils for the study of chemotactic responses are essential in understanding the mechanisms involved in tissue eosinophilia. OBJECTIVE: We compared LTB4 and PAF-induced chemotactic responses of eosinophils isolated by the standard Percoll (positive selection) versus the magnetic cell separation systems (MACS) (negative selection) technique. METHODS: Discontinuous Percoll gradients were preceded by dextran and Ficoll-Paque steps, and followed by gelatin wash and red blood cell (RBC) lysis. MACS isolation included Percoll 1.090 g/mL layering and RBC lysis; incubation with CD16 antibody conjugated to magnetic beads (to bind neutrophils); and isolation of eluate from column positioned in magnet. RESULTS: Percoll-isolated eosinophils migrated to the lipid mediators, LTB4 and PAF, in a dose-responsive fashion. Although MACS isolation provided a greater number and higher purity of eosinophils, these eosinophils migrated less to LTB4 and PAF. Neither dextran sedimentation, dextran and Ficoll-Paque, nor dextran Ficoll-Paque and Percoll prior to MACS isolation reversed the decreased chemotactic responses observed with MACS isolated eosinophils. Further, Percoll-isolated eosinophils further purified with CD16 MicroBeads did not respond as well to LTB4 or PAF. CONCLUSIONS: The technique used to isolate eosinophils clearly affects the chemotactic responsiveness of this cell to LTB4 and PAF. Since several in vivo studies suggest that LTB4 and PAF are eosinophil chemoattractants, Percoll isolation of these cells might be more appropriate for studies involving eosinophil chemotactic responses to these lipid mediators.

Impaired anticoagulant response to infusion of thrombin in atherosclerotic monkeys associated with acquired defects in the protein C system.

To examine the effects of atherosclerosis on the protein C anticoagulant pathway in vivo, we measured anticoagulant responses to intravenous administration of human alpha-thrombin or activated protein C (APC) in cynomolgus monkeys. Two groups of monkeys were fed either a control diet (n=18) or an atherogenic diet (n=12) that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. A third group (n=8) was fed an atherogenic diet for 15 months, and then fed the atherogenic diet supplemented with B vitamins for 6 months to correct the hyperhomocyst(e)inemia. The plasma homocyst(e)ine level was higher in monkeys fed the atherogenic diet (9.6+/-1.0 micromol/L) than in monkeys fed the control diet (3.7+/-0.2 micromol/L) or the atherogenic diet with B vitamins (3.6+/-0.2 micromol/L) (P<0.001). Infusion of thrombin produced a much greater prolongation of the activated partial thromboplastin time in monkeys fed the control diet (52+/-10 seconds) than in monkeys fed the atherogenic diet either with (24+/-4 seconds) or without (27+/-5 seconds) supplemental B vitamins (P<0.02). Thrombin-dependent generation of circulating APC was higher in control (294+/-17 U/mL) than in atherosclerotic (240+/-14 U/mL) monkeys (P<0.05), although levels of fibrinogen, plasminogen, D-dimer, and thrombin-antithrombin complexes were similar in each group. Injection of human APC produced a similar prolongation of the activated partial thromboplastin time in control (31+/-3 seconds) and atherosclerotic (29+/-2 seconds) monkeys. These findings provide evidence for impaired anticoagulation, due partly to decreased formation of APC, in atherosclerosis. The blunted anticoagulant response to thrombin in hypercholesterolemic monkeys was not corrected by supplementation with B vitamins.

Keratinocyte-specific expression of human thrombomodulin in transgenic mice: effects on epidermal differentiation and cutaneous wound healing.

BACKGROUND: Thrombomodulin is a cell-surface glycoprotein that regulates coagulation and fibrinolysis. Expression of thrombomodulin by epidermal keratinocytes is tightly regulated during squamous differentiation and cutaneous wound healing. METHODS: To determine the consequences of overexpression of thrombomodulin on squamous differentiation and wound healing in vivo, we expressed full-length human thrombomodulin in transgenic mice using the human keratin 14 promoter. Human thrombomodulin was detected in keratinocytes of transgenic mice by immunohistochemistry and protein C activation assays. Full-thickness cutaneous wounds were created on the dorsum of transgenic mice and nontransgenic littermates, and allowed to heal for up to 35 days. RESULTS: Transgenic mice had normal viability and appeared healthy up to one year of age. In the skin, human thrombomodulin was expressed in basal and suprabasal keratinocytes, with variable expression in the outer root sheath of hair follicles. Thrombomodulin activity in neonatal epidermis was 2.5- to 3-fold higher in transgenic mice than in nontransgenic littermates (p < 0.01). In cutaneous wounds, human thrombomodulin was expressed in migrating neoepidermal keratinocytes. No differences in keratinocyte migration or re-epithelialization were observed between transgenic and nontransgenic mice, but transgenic mice exhibited delayed collagen bundle deposition within the wound matrix. CONCLUSIONS: These findings demonstrate that keratinocyte thrombomodulin supports activation of protein C, and that thrombomodulin activity in epidermis can be increased by keratinocyte-specific expression of human thrombomodulin in transgenic mice. Expression of human thrombomodulin in keratinocytes does not impair normal squamous differentiation or re-epithelialization of cutaneous wounds, but may modulate collagen reconstitution of the wound matrix.

Interleukin-8 plays a significant role in IgE-mediated lung inflammation.

Interleukin (IL)-8 is a potentially important cytokine in allergic respiratory responses since it is released by many resident lung cells, and it is a potent granulocyte chemoattractant. Therefore, we induced an immunoglobulin (Ig)E-mediated response in human lung samples and studied whether IL-8 was produced in sufficient quantities to promote human neutrophil and eosinophil migration across naked filters and endothelial and pulmonary epithelial monolayers cultured on these filters. Fresh human lung fragments from 16 thoracotomy specimens were treated with either a 1:100 dilution of anti-IgE or buffer (control) for 30 min. All anti-IgE treated lung samples had significant release of histamine and neutrophil and eosinophil chemotactic activity. Fourteen of the 16 lung samples had a significant increase in IL-8 subsequent to anti-IgE treatment (p<0.01). Anti-IL-8 antibody (4 microg x mL[-1]) inhibited 42% and 53% of neutrophil and eosinophil chemotactic activity respectively, contained in supernatants from anti-IgE-treated lung samples. Finally, we found that IL-8 at a concentration near that measured after anti-IgE treatment of lung samples (2,000 pg x mL[-1]) induced neutrophil and eosinophil migration through naked filters and endothelial and pulmonary epithelial cell monolayers. Thus, human lung IgE-mediated responses in vitro results in the rapid release of interleukin-8 in amounts sufficient to affect a biological response, granulocyte transcellular migration, indicating that interleukin-8 may play a significant role in allergic respiratory diseases.

Tumor necrosis factor alpha is necessary for granulocyte-macrophage-colony-stimulating-factor-induced eosinophil transendothelial migration.

BACKGROUND: We have previously shown that granulocyte macrophage-colony stimulating factor (GM-CSF) was capable of inducing eosinophil migration across naked filters but not endothelial monolayers. Tumor necrosis factor alpha (TNF-alpha) has been shown to be a key factor in granulocyte adhesion and transendothelial migration. METHODS: We, therefore, pretreated human umbilical vein endothelial cell (HUVEC) monolayers with TNF-alpha and studied whether TNF-alpha could support GM-CSF-induced eosinophil transendothelial migration. RESULTS: We found that TNF-alpha supported GM-CSF-induced eosinophil transendothelial migration and that this process was: (1) dependent upon GM-CSF and TNF-alpha dose; (2) time-dependent; (3) not due to TNF-alpha having a chemotactic effect itself; (4) not due to TNF-alpha-induced soluble factor production by endothelium, and (5) inhibitable by actinomycin D. We next studied the specificity of this response. Neutrophils did not migrate across TNF-alpha-pretreated endothelium in response to GM-CSF. TNF-alpha pretreatment of A549 human type-II-like epithelial lung cells (A549) did not support GM-CSF-induced transepithelial migration. Neither interleukin (IL)-1 nor GM-CSF pretreatment of the HUVEC supported GM-CSF-induced transendothelial migration. However, IL-5 induced eosinophil migration through naked filters as well as TNF-alpha-pretreated HUVEC in a manner analogous to GM-CSF. Antibodies to ICAM-1, but not VCAM-1 significantly inhibited this response. Although IL-1 did not support GM-CSF-induced eosinophil transendothelial migration, IL-1 and TNF-alpha induced equivalent expression of ICAM-1 on HUVEC. CONCLUSION: Thus, TNF-alpha-supported eosinophil transendothelial migration in response to GM-CSF (and IL-5) is dependent upon ICAM-1, and is both specific and complex.

Functional interactions between the thrombin receptor and the T-cell antigen receptor in human T-cell lines.

The proteolytically activated thrombin receptor (TR) is expressed by T lymphocytes, which suggests that thrombin may modulate T-cell activation at sites of hemostatic stress. We examined the relationship between TR function and T-cell activation in the Jurkat human T-cell line and in T-cell lines with defined defects in T-cell antigen receptor (TCR) function. Stimulation with thrombin or the synthetic TR peptide SFLLRN produced intracellular Ca2+ transients in Jurkat cells. As the concentration of TR agonist was increased, peak Ca2+ mobilization increased, but influx of extracellular Ca2+ decreased. TR signaling was enhanced in a TCR-negative Jurkat line and in T-cell lines deficient in the tyrosine kinase lck or the tyrosine phosphatase CD45, both of which are essential for normal TCR function. TCR cross-linking with anti-CD3 IgM desensitized TR signaling in Jurkat cells, but not in CD45-deficient cells. A proteinase-activated receptor (PAR-2)-specific agonist peptide, SLIGKV, produced small Ca2+ transients in both MEG-01 human megakaryocytic cells and Jurkat cells, but was less potent than the TR-specific agonist TFRIFD in both cell types. Like TR signaling, PAR-2 signaling was enhanced in TCR-negative or lck-deficient Jurkat clones. These findings provide evidence for functional cross-talk between proteolytically activated receptors and the TCR.

Human lung anaphylaxis results in rapid release of interleukin-4.

BACKGROUND: Interleukin-4 has been implicated as having numerous roles in the inflammatory responses characteristic of allergic asthma. Interleukin-4 has been shown to be involved in IgE synthesis, upregulation of BCAM-1 on endothelium, and promotion of inflammatory cell infiltration into the airways. OBJECTIVE: We therefore examined whether IL-4 was produced after an IgE-mediated response in human lung samples. RESULTS: Anti-IgE treatment of 12 human lungs resulted in the significant release of IL-4 within 30 minutes. CONCLUSIONS: Although the source of released IL-4 is unknown, the rapid release of IL-4 suggests that cells with performed stores, such as mast cells and eosinophils, are involved. Once released, IL-4 may play an important role in the pathogenesis of asthma.

Isolation technique alters eosinophil migration response to IL-8.

Disparate reports exist on the eosinophil chemotactic capacity of interleukin-8 (IL-8). We hypothesized that the difference is due to the methods used to purify eosinophils. We therefore compared the eosinophilotactic capacity of IL-8 on human cells isolated by Percoll (positive selection) vs. magnetic cell separation system (MACS) (negative selection). Discontinuous Percoll gradients were preceded by dextran and Ficoll-Paque steps, and followed by gelatin wash and red blood cell (RBC) lysis. MACS isolation included: Percoll 1.090 g/ml layering and RBC lysis; incubation with CD16 antibody conjugated to magnetic beads (to bind neutrophils); and isolation of eluate from column positioned in magnet. Percoll isolated eosinophils migrated to IL-8 in a dose-responsive fashion. Although MACS isolation provided a greater number and higher purity of eosinophils, these eosinophils did not migrate to IL-8. Neither dextran sedimentation, Ficoll-Paque and Percoll prior to, nor Percoll discontinuous gradients subsequent to, MACS isolation reversed the negative chemotactic response. Moreover, Percoll-isolated eosinophils further purified with CD16 MicroBeads no longer chemotactically responded to IL-8. This inhibition was not due to change in eosinophil purity, a loss of eosinophil adhesion molecules or activation markers, the presence of a soluble neutrophil or eosinophil inhibitor or the effect of the magnet. Thus, the technique used to isolate eosinophils clearly affects the chemotactic responsiveness of this cell to IL-8. Since several in vivo studies suggest that IL-8 is an eosinophil chemoattractant, Percoll isolation of these cells might be more appropriate for studies involving eosinophil chemotactic responses to IL-8.

Eosinophil migration in response to three molecular species of platelet activating factor.

Multiple molecular species of the eosinophil chemoattractant platelet activating factor (PAF) are produced as a result of inflammatory processes. We therefore compared the ability of three naturally occurring PAF species (C16:0, C18:0, and C18:1), which only varied at carbon 1, to induce eosinophil chemotaxis through naked 3-microns pore polycarbonate filters. Timecourse experiments indicated that all species of PAF tested induced significant and equivalent eosinophil migration at 1 h which peaked at 2 h. Overall, the rank order of chemotactic potency for the PAF species was relatively equivalent. The specific PAF antagonist WEB 2086 inhibited eosinophil migration induced by all three PAF species equally. We conclude that the degree of PAF-induced eosinophil migration is not dependent upon the molecular species of PAF.

Cytokines induce selective granulocyte chemotactic responses.

Neutrophils, eosinophils and cytokines are important in allergic airway inflammatory responses. However, it is unclear how cytokines selectively influence neutrophils versus eosinophils to migrate to an inflammatory site. The cytokines, transforming growth factor-beta1 (TGF-beta1), interleukin (IL)-1alpha, IL-5, IL-8, granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha), are released subsequent to allergic reactions and affect both neutrophil and eosinophil functions. We studied whether these cytokines differed in capacity to induce human neutrophil versus eosinophil migration through naked filters and human umbilical vein endothelial cell (HUVEC) and human pulmonary type II-like epithelial (A549) cell monolayers grown on filters. Dose-response experiments using all barriers were performed for each granulocyte and cytokine. TGF-beta1 did not induce granulocyte migration. IL-5 induced eosinophil migration only through naked filters. IL-1alpha stimulated neutrophil migration through cellular barriers, but not through naked filters. TNF-alpha and GM-CSF induced neutrophil and eosinophil migration through filters, but only neutrophil migration through cellular monolayers. Only IL-8 induced significant neutrophil and eosinophil migration; however, there were clear-cut differences between the neutrophilotactic and eosinophilotactic responses through all barriers employed. Thus, our data show that these cytokines induce distinct chemotactic responses for neutrophils versus eosinophils. Moreover, by using relevant cellular barriers versus naked filters, our data better examines the capability of these cytokines to induce selective granulocyte migration to an inflammatory site in lung diseases such as asthma.

Comparative studies indicate that platelet-activating factor is a relatively weak eosinophilotactic mediator.

Eosinophils are important immune effector cells in a variety of allergic responses and inflammatory lung diseases. Bacterial products and inflammatory mediators have been implicated in inducing an influx of eosinophils into the respiratory tract subsequent to an acute inflammatory response. Therefore, to better understand the role of eosinophils in lung inflammation, we compared the ability of three known chemoattractants, formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and platelet-activating factor (PAF), to induce human eosinophils to migrate across 3.0-microns-pore naked filters and human umbilical vein endothelial cells (HUVEC) and A549 human pulmonary type II-like epithelial (A549) cells cultured in monolayers on these filters. Kinetic experiments indicated that eosinophil migration through all three barriers occurred by 60 min and plateaued by 2 h. Each of these chemoattractants induced eosinophil migration in dose-responsive fashion across all three barriers. Although similar maximal eosinophil migration was observed, the doses at which this occurred varied, indicating that the rank order of potency through naked filters is FMLP > PAF > or = LTB4. However, their relative chemotactic potency through cellular barriers was different, with FMLP > LTB4 > PAF. In contrast to previous studies with neutrophils, the rank order of potency of the three chemoattractants was not influenced by the barrier through which the eosinophil migrated. Thus, these and previous data show that FMLP, LTB4, and PAF are eosinophil and neutrophil chemoattractants. Therefore, it is likely that these three agents are important mediators of granulocytic inflammatory responses in the lung, albeit with different potency profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-8 is a potent mediator of eosinophil chemotaxis through endothelium and epithelium.

Interleukin-8 (IL-8), a potent pro-inflammatory cytokine, has been shown to have chemotactic activity for neutrophils, lymphocytes, and basophils. Effects of IL-8 on eosinophil chemotaxis are unresolved. Because eosinophils accumulate at the site of allergic inflammation and may play a role in the pathogenesis of asthma, we investigated the eosinophilotactic capacity of IL-8. We examined the ability of IL-8 to induce human eosinophil migration across 3-microns pore naked filters, and human umbilical vein endothelial cell and human pulmonary type II-like epithelial cell (A549) monolayers cultured on these filters. IL-8 induced similar dose-related eosinophil migration through all three barriers. Kinetic experiments indicated more rapid migration through noncellular barriers but equivalent migration through all barriers by 3 h. Chemotactic/chemokinetic data show that IL-8-induced eosinophil migration is chemotactic. We also determined that the ability of IL-8 to induce transcellular migration was unique in comparison with other cytokines and was not dependent on the use of fresh vs. passaged monolayer cells as barriers. Therefore our data indicate that IL-8 may play a significant role in tissue eosinophilia observed in allergic respiratory diseases.

Platelet-activating factor-induced human eosinophil transendothelial migration: evidence for a dynamic role of the endothelium.

Stimulated migration of eosinophils out of the bloodstream and into the lung is key in the development of tissue eosinophilia and inflammation in asthma. Platelet-activating factor (PAF) has been implicated as an important inflammatory mediator in asthma pathogenesis in part because of its chemotactic capacity. We therefore studied the ability of PAF to induce human peripheral blood eosinophil migration through naked filters and human umbilical vein endothelial cells (HUVECs) cultured on these filters. PAF induced eosinophil migration through both barriers in a time-dependent fashion, with maximal eosinophil migration occurring at 180 min. Significant eosinophil migration was observed at PAF concentration > or = 0.1 microM and was dose dependent up to 10.0 microM. No significant differences in eosinophil chemotactic responses were noted between naked filter and HUVEC barriers. The PAF receptor antagonist, WEB 2086, inhibited (> 85%) eosinophil transendothelial migration when co-incubated with PAF or when used as a pretreatment of either the eosinophils or HUVECs. However, WEB 2086 pretreatment of HUVECs did not inhibit PAF-induced neutrophil transendothelial migration, nor did it affect leukotriene B4-induced neutrophil or eosinophil transendothelial migration. Thus, the data indicate that the endothelial cell plays an important role in PAF-induced eosinophil inflammatory processes. Moreover, these data suggest that PAF's pathogenic role in asthma may in part be due to its ability to stimulate eosinophil migration across endothelial barriers and into the airways.

Comparison of methodologies to measure human lung histamine.

The measurement of histamine in samples obtained from human lung is important in determining the roles of histamine and mast cells in normal and disease states. We, therefore, compared different assays for the measurement of histamine in human lung samples. Both a single isotope enzymatic assay and a radioimmunoassay (RIA) were capable of accurately measuring the low concentrations of histamine (0.05-2.0 ng/ml) normally found in bronchoalveolar lavage fluid. The RIA was also able to measure histamine levels up to 1500 ng/ml in human lung tissue samples. Moreover, the RIA measurement of high levels of histamine in lung samples compared favorably to an automated spectrofluorometric method. Unlike either the single isotope enzymatic assay or the automated spectrofluorometric assay which have effective capabilities at less than and greater than 2 ng/ml, respectively, the RIA can accurately measure histamine levels from 0.05 to 1500 ng/ml. Since the RIA is easier to perform, less costly, and has a wider range of effectiveness, this assay should prove valuable in assessing histamine levels from a variety of human lung samples, thereby, providing an avenue to elucidate the roles of histamine and mast cells in lung functions.

The incidence of a new human cross-reactive idiotype linked to subgroup VHIII heavy chains.

Cross-reactive idiotypes (CRI) on human rheumatoid factors (RF), which are identified by murine monoclonal antibodies (mAb), have proved useful in defining both the incidence and the structural characteristics of these autoantibodies. In this study, a new murine anti-idiotypic reagent, mAb B6, has been used to identify and define the expression of a distinct heavy chain CRI. The B6 CRI was found on 20% of monoclonal IgM (16 of 81), but on only 5% of monoclonal IgA (1 of 20) and on no monoclonal IgG. In addition, this CRI was expressed exclusively on a subset of Ig derived from the VHIII protein variable region subgroup. In immunoblotting experiments, the mAb B6 bound directly to the heavy (H) chains of CRI positive proteins. The B6 CRI was found frequently on monoclonal IgM-RF molecules, and the mAb B6 could inhibit the binding of the RF to its IgG antigen. It was also demonstrated that Staphylococcus aureus protein A (SpA), which has recently been shown to bind to the F(ab) region of VHIII molecules, could block the interaction of some B6 CRI positive IgM to the anti-CRI. These experiments suggest that the B6 CRI is a marker for one or a few VHIII genes and that it is expressed commonly on IgM paraproteins, many of which have RF activity.

A highly informative probe for two polymorphic Vh gene regions that contain one or more autoantibody-associated Vh genes.

Efforts to determine the role of specific Ig variable region (V) genes in human autoimmune responses have been hampered by the lack of suitably polymorphic probes. Recently we isolated a heavy chain V (Vh) gene, designated Humhv3005, that is 99% homologous to the 1.9III Vh gene and can encode an anti-DNA antibody. To study the relation between these two genes, different DNA fragments from the isolated Humhv3005 clone were used to probe Southern blots of human genomic DNA. A 1.6-kb Eco RI fragment (designated hv3005/E1.6) was found to hybridize with only one band in Eco RI-digested DNA, and with two major bands in Bam HI-digested DNA. Importantly, the sizes of the latter two bands were indistinguishable from the corresponding Bam HI fragment sizes of the isolated hv3005 clone and the isolated 1.9III clone, respectively. Population and family studies with the hv3005/E1.6 probe revealed five different hybridization patterns of these two characteristic bands, which defined nine possible genotypes for two human Ig Vh gene loci. Together the data demonstrate that hv3005/E1.6 is a highly informative probe for an autoantibody-associated Vh gene(s), and should prove useful in elucidating the role of Ig Vh genes in autoimmune diseases.