Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient

OBJECTIVE: To investigate the production of proinflammatory cytokines and expression of cell adhesion molecules in the rheumatoid nodule. METHODS: Cytokine content (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], and IL-1 receptor antagonist [IL-1Ra]), at the messenger RNA (mRNA) and protein levels, and cell adhesion molecule expression were studied in 16 rheumatoid nodules and 6 synovial membranes. RESULTS: Macrophages in the rheumatoid nodules contained TNFalpha, IL-1beta, and IL-1Ra mRNA and protein, particularly in perivascular cells of the stroma and in the palisading layer. All cell adhesion molecules studied were expressed in both the rheumatoid nodules and synovial membranes, with increased expression of E-selectin in the rheumatoid nodule compared with the synovial membrane, and with the absence of vascular cell adhesion molecule 1 expression on cells of the palisading layer in the rheumatoid nodule. CONCLUSION: The presence of similar proinflammatory cytokines and cell adhesion molecules in the rheumatoid nodule and synovial membrane suggests that similar pathogenic processes result in the chronic inflammation and tissue destruction in these lesions.     

Endothelial cell activation by sera containing HLA antibodies is mediated by interleukin-1

BACKGROUND: It has been suggested that antibodies which are associated with chronic pathological conditions such as chronic rejection and autoimmune diseases have the capacity to activate endothelial cells by induction and up-regulation of adhesion molecules. It has also been suggested that HLA antibodies formed by patients awaiting transplantation can activate endothelial cells. These antibodies include HLA and those that bind to endothelial cells. METHODS: We have further investigated this phenomenon using monoclonal antibodies against HLA class I determinants and sera from aortic valve graft recipients, containing strong HLA antibodies. The effect of 24-hr incubation of antibodies/serum with human umbilical vein endothelial cells (HUVECs) on adhesion molecule expression was measured by flow cytometry. RESULTS: HLA monoclonal antibodies had no effect on ICAM-1 expression on HUVECs. Five of 31 (16%) patients' sera caused strong up-regulation of adhesion molecules (ICAM-1, vascular cell adhesion molecule-1, and E-selectin) but this did not correlate with HLA specificity, IgG, or IgM binding to HUVECs. The activity, found in whole serum and IgG-depleted fractions was inhibited by neutralizing antibodies against interleukin (IL)-1beta and tumor necrosis factor-alpha. Examination of patient sera for presence of IL-1beta demonstrated high levels of IL-1beta in all five sera (range, 30 -500 U/ml) as well as in samples from an additional three patients. CONCLUSION: The ability to activate endothelial cells detected in our patient sera was caused by cytokines and not antibody. Our observation that addition of cytokines to sera before separation into large and low molecular weight fractions demonstrated retention of cytokines in both fractions may be a confounding issue when investigating endothelial cell activation by patients' sera.     

Chemokine and cell adhesion molecule mRNA expression and neutrophil infiltration in lipopolysaccharide-induced hepatitis in ethanol-fed rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.     

Mechanisms of eosinophil recruitment

As with other types of leukocytes, mechanisms that function to enable the recruitment of eosinophils into specific sites of immune reactions involve a complex and cumulative interplay of many molecules and pathways. No single chemoattractant is specific for eosinophils, but rather various chemoattractants active on eosinophils can also elicit migration of other specific cell types. Humoral mediators causing eosinophil migration include C5a and platelet-activating factor, whereas cytokines active as eosinophil chemoattractants include interleukin (IL)-2, IL-3, IL-5, granulocyte/macrophage colony-stimulating factor, lymphocyte chemoattractant factor, and RANTES. Eosinophils utilize several pathways to adhere to vascular endothelial cells, including binding to intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). The lack of binding of neutrophils to VCAM-1 and the enhanced expression of VCAM-1 elicited by IL-4 contribute to preferential eosinophil accumulation. Eosinophil recruitment is dependent not only on ligands expressed on eosinophils and molecules inducible on endothelial cells but also on processes active during transendothelial migration and extravascular migration in the extracellular spaces.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.     

Inflammation and glial responses in ischemic brain lesions

Focal cerebral ischemia elicits a strong inflammatory response involving early recruitment of granulocytes and delayed infiltration of ischemic areas and the boundary zones by T cells and macrophages. Infiltration of hematogenous leukocytes is facilitated by an upregulation of the cellular adhesion molecules P-selectin, intercellular adhesion molecule-1 and vascular adhesion molecule-1 on endothelial cells. Blocking of the leukocyte/endothelial cell adhesion process significantly reduces stroke volume after transient, but not permanent middle cerebral artery occlusion. In the infarct region microglia are activated within hours and within days transform into phagocytes. Astrocytes upregulate intermediate filaments, synthesize neurotrophins and form glial scars. Local microglia and infiltrating macrophages demarcate infarcts and rapidly remove debris. Remote from the lesion no cellular infiltration occurs, but astroglia and microglia are transiently activated. Astrocytic activation is induced by spreading depression. In focal ischemia neurons die acutely by necrosis and in a delayed fashion by programmed cell death, apoptosis. Proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta are upregulated within hours in ischemic brain lesions. Either directly or via induction of neurotoxic mediators such as nitric oxide, cytokines may contribute to infarct progression in the post-ischemic period. On the other hand, inflammation is tightly linked with rapid removal of debris and repair processes. At present it is unclear whether detrimental effects of inflammation outweigh neuroprotective mechanisms or vice versa. In global ischemia inflammatory responses are limited, but micro- and astroglia are also strongly activated. Glial responses significantly differ between brain regions with selective neuronal death and neighbouring areas that are more resistent to ischemic damage.     

Observation of the nuCH Excited Vibrational Levels in the ?1A" State of HCP by IR-UV Double Resonance Spectroscopy

Expression of membrane-bound (mb) and soluble (s) forms of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-alpha (TNF-alpha) has been measured by enzyme-linked immunosorbent assay in cultured human brain microvessel endothelial cells. Both the mb and the s forms of VCAM-1 and ICAM-1 were upregulated by TNF-alpha; however, the stimulation of the s forms was delayed in time. When piracetam, a neuroprotective drug, was added to the tissue culture medium simultaneously with TNF-alpha, the expression of mbVCAM-1 and ICAM-1 was lowered. Differential upregulation of mb and s forms of adhesion molecules and a novel effect of piracetam have been demonstrated in human brain microvessel endothelial cell cultures.     

Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in human endothelial cells mediates alpha 2,6-sialylation of adhesion molecules and CD22 ligands

Sialic acids decorating blood and cell surface proteins can play important roles in various biological processes. The inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1, as well as bacterial lipopolysaccharide, can activate vascular endothelium, increasing expression of several surface glycoproteins. Here we show that treatment of cultured human endothelial cells (HEC) with TNF-alpha, interleukin-1, or lipopolysaccharide causes increased expression of the enzyme beta-galactoside alpha-2,6-sialytransferase (alpha 2-6STN). TNF-alpha was most effective, inducing a 3.5-fold enhancement of cell-associated sialytransferase activity by 72 h. In addition, activated HEC secreted a large portion of the induced sialyltransferase activity into the medium. Analysis of labeled HEC showed both a relative and an absolute increase of alpha 2,6-linked sialic acid on N-linked oligosaccharides after TNF-alpha stimulation. This coincided with increased expression of endothelial glycoproteins bearing N-linked glycans with alpha 2,6-linked sialic acid detected by the lectin Sambucus nigra agglutinin. The cytokine-inducible endothelial cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 are among these glycoprotein substrates for alpha 2-6STN. These changes also correlated with a substantial increase in binding sites for CD22 beta, a mammalian lectin known to recognize oligosaccharides carrying multiple copies of alpha 2,6-linked sialic acid. Northern analysis revealed increased levels of mRNA encoding alpha 2-6STN. Thus, activation of endothelial cells during inflammatory and immunological processes may induce alpha 2-6STN, which can participate in sialylation of other activation-dependent molecules.     

Expression of the beta 7 integrin by human endothelial cells

Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells.     

Endothelial cells in culture: an experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [3H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor-alpha and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selection and vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also in rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.     

International study to compare antigen-specific methods used for the measurement of antiplatelet autoantibodies

BACKGROUND/AIMS: Cell adhesion phenomena are relevant in the immune mechanisms leading to organ damage in various diseases. Patients with alcoholic cirrhosis present with immune alterations that include findings of immunodeficiency and indications of an activated immune response. METHODS: In 37 patients with alcoholic cirrhosis we have determined the expression of surface antigens and adhesion molecules on peripheral lymphocytes and monocytes, serum levels of immunoglobulins, circulating cytokines, namely tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta, serum soluble intercellular adhesion molecule and neopterin. RESULTS: In patients, we found an increased expression of several adhesion molecules ICAM-1, LFA-3 and MAC-1 in lymphocytes, LFA-3 in monocytes and surface activation markers CD71 and DR in lymphocytes, as well as increased concentrations of the serum parameters measured: IgA, IgG, IgM, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, soluble ICAM-1 and neopterin, in comparison with controls. CONCLUSIONS: The enhancement of the adhesion phenomena in circulating mononuclear cells of patients with cirrhosis correlates to the severity of the disease and is related to other parameters of immune activation.     

Cell adhesion molecules--update

Cell adhesion molecules are glycoproteins expressed on the cell surface and play an important role in inflammatory as well as neoplastic diseases. There are four main groups: the integrin family, the immunoglobulin superfamily, selectins, and cadherins. The integrin family has eight subfamilies, designated as beta 1 through beta 8. The most widely studied subfamilies are beta 1 (CD29, very late activation [VLA] members), beta 2 (leukocyte integrins such as CD11a/CD18, CD11b/CD18, CD11c/CD18, and alpha d beta 2), beta 3 (CD61, cytoadhesions), and beta 7 (alpha 4 beta 7 and alpha E beta 7). The immunoglobulin superfamily includes leukocyte function antigen-2 (LFA-2 or CD2), leukocyte function antigen-3 (LFA-3 or CD58), intercellular adhesion molecules (ICAMs), vascular adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The selectin family includes E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). Cadherins are major cell-cell adhesion molecules and include epithelial (E), placental (P), and neural (N) subclasses. The binding sites (ligands/receptors) are different for each of these cell adhesion molecules (e.g., ICAM binds to CD11/CD18; VCAM-1 binds to VLA-4). The specific cell adhesion molecules and their ligands that may be involved in pathologic conditions and potential therapeutic strategies by modulating the expression of these molecules will be discussed.