Analysis of cytokine mRNA and protein in rheumatoid arthritis tissue revealed that many proinflammatory cytokines such as TNF alpha, IL-1, IL-6, GM-CSF, and chemokines such as IL-8 are abundant in all patients regardless of therapy. This is compensated to some degree by the increased production of anti-inflammatory cytokines such as IL-10 and TGF beta and cytokine inhibitors such as IL-1ra and soluble TNF-R. However, this upregulation in homeostatic regulatory mechanisms is not sufficient as these are unable to neutralize all the TNF alpha and IL-1 produced. In rheumatoid joint cell cultures that spontaneously produce IL-1, TNF alpha was the major dominant regulator of IL-1. Subsequently, other proinflammatory cytokines were also inhibited if TNF alpha was neutralized, leading to the new concept that the proinflammatory cytokines were linked in a network with TNF alpha at its apex. This led to the hypothesis that TNF alpha was of major importance in rheumatoid arthritis and was a therapeutic target. This hypothesis has been successfully tested in animal models, of, for example, collagen-induced arthritis, and these studies have provided the rationale for clinical trials of anti-TNF alpha therapy in patients with long-standing rheumatoid arthritis. Several clinical trials using a chimeric anti-TNF alpha antibody have shown marked clinical benefit, verifying the hypothesis that TNF alpha is of major importance in rheumatoid arthritis. Retreatment studies have also shown benefit in repeated relapses, indicating that the disease remains TNF alpha dependent. Overall these studies demonstrate that analysis of cytokine expression and regulation may yield effective therapeutic targets in inflammatory disease.

Role of cytokines in rheumatoid arthritis

It is increasingly apparent that not only is a cure for the current worldwide diabetes epidemic required, but also for its major complications, affecting both small and large blood vessels. These complications occur in the majority of individuals with both type 1 and type 2 diabetes. Among the most prevalent microvascular complications are kidney disease, blindness, and amputations, with current therapies only slowing disease progression. Impaired kidney function, exhibited as a reduced glomerular filtration rate, is also a major risk factor for macrovascular complications, such as heart attacks and strokes. There have been a large number of new therapies tested in clinical trials for diabetic complications, with, in general, rather disappointing results. Indeed, it remains to be fully defined as to which pathways in diabetic complications are essentially protective rather than pathological, in terms of their effects on the underlying disease process. Furthermore, seemingly independent pathways are also showing significant interactions with each other to exacerbate pathology. Interestingly, some of these pathways may not only play key roles in complications but also in the development of diabetes per se. This review aims to comprehensively discuss the well validated, as well as putative mechanisms involved in the development of diabetic complications. In addition, new fields of research, which warrant further investigation as potential therapeutic targets of the future, will be highlighted.

Mechanisms of Diabetic Complications

https://www.cell.com/immunology/pdf/0167-5699(93)90267-O.pdf

Circulating adhesion molecules in disease.

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(97)09032-6.pdf

Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men

Adhesion molecules and atherosclerosis

Endothelial cells respond to several cytokines by a rapid increase in expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1), followed by a gradual decline. The fate of these molecules, which was so far unknown, was studied. Specific sandwich ELISA for the detection of soluble (s)E-selectin and sICAM-1 were developed. In supernatant, centrifuged 3 hr at 100,000 g to remove microparticles, from human umbilical vein endothelial cells (HUVEC) activated with tumour necrosis factor (TNF), interleukin-1 (IL-1) or lipopolysaccharide (LPS), E-selectin and ICAM-1 molecules could be detected. Biochemical analysis revealed that sE-selectin migrated as a band of approximately 94,000 MW. The amount of soluble adhesion molecules released was directly correlated with cell surface expression. Maximal release of E-selectin was observed 6-12 hr after activation of HUVEC and decreased to below detection limit 24 hr after activation. After activation, release of ICAM-1 gradually increased with ICAM-1 cell surface expression, and reached a plateau after 24 hr, which was constant for 3 days. Since E-selectin and ICAM-1 are highly expressed at inflammatory sites, the resulting high concentrations of released E-selectin and ICAM-1 may affect interactions of leucocytes with endothelial cells. The physiological role, however, of the release of E-selectin and ICAM-1 remains to be elucidated.

E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.

Evidence for endocytosis of E-selectin in human endothelial cells

We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.

A Comparison of Substrates for Human Umbilical Vein Endothelial Cell Culture

E-selectin (ELAM-1) is an adhesion molecule for leukocytes that is transiently expressed on endothelial cells. Following cell surface expression of E-selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor, the induced E-selectin molecules are rapidly degraded. The kinetics of turnover of surface disposed E-selectin were investigated. The rapid disappearance of surface E-selectin is temperature dependent and sensitive to the lysosomotropic agent chloroquine. The half-life of E-selectin is not affected by inclusion of soluble sialyl Lewis x (sLex) ligands in the medium. Surface E-selectin is phosphorylated on one or more serine residues, but this modification is not obviously related to internalization.

E. F. Smeets

Papers

E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

Evidence for endocytosis of E-selectin in human endothelial cells

A Comparison of Substrates for Human Umbilical Vein Endothelial Cell Culture

Phosphorylation of surface E-selectin and the effect of soluble ligand (Sialyl Lewisx) on the half-life of E-selectin