Tumor necrosis factor alpha induction in human monocytes.

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.

Platelet interaction with [125I]-labeled collagen type III: requirement of fibrillar structure formation.

Interaction of collagen type III (CIII) with washed human platelets was studied using CIII preparation from human placenta. CIII was labeled with [125I] and [125I]-CIII in monomeric and fibrillar form [( 125I]-CIIIm and [125I]-CIIIf respectively) were incubated with platelets at room temperature. Platelet-associated and free labels were separated by centrifugation through 20% sucrose. Binding of [125I]-CIIIf was unsaturable, linearly dependent on the concentration of label and represented 28 +/- 3% of the added protein. In comparison with CIIIf, binding of [125I]-CIIIm was minimal and represents only 0.9 +/- 0.2% of the added protein. The binding of [125]-CIIIm was also nonsaturable and linearly depend on the concentration of the labeled protein. Platelet activation neither increases the CIIIf binding, nor stimulates the binding of CIIIm. The binding of [125I]-CIIIf was not inhibited by the excess of the unlabeled CIIIm. The data obtained suggests the absence of high-affinity collagen receptors in platelets and corroborates the hypothesis of multiple low-affinity interactions between collagen fibrils and platelet surface. Binding of CIIIf was very fast--the level of binding reached a plateau within 1 min, and was similar in the presence of Ca2+/Mg2+ and EDTA. Spectrophotometrically undetectable microfibril formation during the lag phase of fibrillogenesis was sufficient for nearly the same as with large fibrils binding of CIII to platelets. Unlike platelets red blood cells (RBC) fail to bind significant amounts of [125I]-CIIIf.

Participance of fibronectin and various collagen types in the formation of fibrous extracellular matrix in cardiosclerosis.

Investigation of the extracellular matrix composition of the left heart ventricle was carried out on autopsy material of subjects, aged from 60 to 70 years, in a number of cases, including: (1) tissue without cardiosclerosis; (2) granulation tissue formed 2 weeks after infarction; (3) post-infarctial fibrous scars; (4) diffuse cardiosclerosis in consequence of stenotic coronary atherosclerosis. Cryostat sections treated with highly specific antibodies to fibronectin and types I, III, IV and V collagens were examined by the indirect immunofluorescence technique. Fibronectin and the mentioned collagenous proteins were detected in the extracellular matrix of granulation tissue. In contrast, fibronectin and collagen type IV were not revealed in post-infarctial fibrous scars. Collagen types III and V were diffusely distributed in fibrous tissue, whereas collagen type I was demonstrated to accumulate preferentially in the deeper regions of post-infarctial scars. Fibronectin and collagen types I, III, V, but never type IV, were also found in the connective tissue in diffuse cardiosclerosis. The significance of type V collagen in the extracellular matrix is discussed.

In vivo administration of antibodies against type I collagen in rat: the specific accumulation in spleen.

[125I]-labelled rabbit antibodies against rat type I collagen and non-immune IgG were injected into rat circulation. The kinetics of their clearance and the biodistribution in different organs were studied. Both preparations showed very similar clearance rate, the kinetics fitting bi-exponential approximation with characteristic parameters t1,1/2 = 201 +/- 20 min before 320 min and t2,1/2 = 1350 +/- 450 min at times over 320 min for antibodies and 258 +/- 45 min and 890 +/- 140 min for IgG. The specific affinity of the circulating antibodies did not decrease within 24 hours. The antibodies were specifically accumulated in spleen, where their accumulation was 5-fold higher than that of non-immune IgG. Accumulation of antibodies was maximal 3 hours after the injection. The localization ratio (i.e. the ratio of the amount of the antibodies per g of tissue to that per g of blood) reached a maximum 24 hours after the injection and remained stable for 120 hours. Immunofluorescent staining of spleen sections resulted in a bright fluorescence of dense collagenous structures in the trabeculae and in the wall of the central follicular arterium, bright spot fluorescence in the marginal zone of the follicle, and diffuse fluorescence in the red pulp. These findings suggest an unusually high accessibility of collagen type I in spleen to circulating blood plasma components.

Relative distribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intima of human arteries.

Spatial distribution of fibronectin and type I, III, IV and V collagen has been investigated in normal arterial intima, fatty streaks, and atherosclerotic plaques by indirect immunofluorescence on transverse sections. Two distinct types of extracellular matrix were revealed in atherosclerotic lesions. The fibrous plaques consisted mostly of interstitial collagen types I and III, contained moderate amounts of type V and none of type IV collagen or fibronectin. In the extracellular matrix of the fatty streaks and in some areas of the fibrous plaques containing large amounts of subendothelial cells, some interstitial collagen was revealed, an increased amount of type IV, some type V collagen and a lot of fibronectin. Similarities of the extracellular matrix in atherosclerotic lesions and granulation tissues are discussed.

Distribution of type I, III, IV and V collagen in normal and atherosclerotic human arterial wall: immunomorphological characteristics.

35 autopsies--aged 30 to 75 years--were investigated in order to establish trends of collagen localization in various types of arteries depending on age, arterial size and degree of atherosclerosis. Cryostat sections stained with highly specific antibodies to human types I, III, IV or V collagen, or with the antiserum to smooth muscle myosin were examined by the indirect immunofluorescence technique. Localization of type III collagen was very similar to that of type I. Fibrous structures of both type I and type III were then major constituents of the intima, media and adventitia. Sparse fibrils of type I and type III collagens were revealed in the subendothelium of unaffected intima. They gradually became abundant in the deeper intimal layers contrasting with loose fibrillar formations of the media. The content of interstitial collagens was significantly increased in the subendothelium of local intimal thickenings and in a thickened intima of the aged. This fact, considering the thrombogenicity of interstitial collagens, may be relevant to the atherogenesis through the "response-to-injury" mechanism. Type IV and type V collagens are localized to the endothelial basement membrane and basement membranes of smooth muscle cells of the intima and media. Diffusely distributed type V collagen was also observed in the intercellular space of the intima. In lipid streaks, parallel layers of condensed interstitial collagens separated groups of cells and extracellular lipid depositions. In fibrous plaques, types I and III became prevalent structural elements and their densely packed fibers occupied whole regions devoid of any type IV and type V collagen. Heavily thickened type IV collagen structures surrounding individual smooth muscle cells were found in fibrous plaques, but never, in unaffected intima.

Type I and III collagens as a possible target for drug delivery to the injured sites of vascular bed.

Interaction of anti-human collagen types I and III antibodies, as well as human red blood cells conjugated with these antibodies, with the surface of denuded intima of human aorta has been studied. Data on the accessibility of antigenic determinants of collagen types I and III for antibodies and red blood cells conjugated with these antibodies have been obtained in ex vivo experiments in an original model. On the basis of the obtained results it is concluded that antigenic determinants of collagen types I and III exposed as a result of blood vessel wall injury can serve as a target for drug delivery to the injured site(s).