Cytokine gene expression in aortic adventitial inflammation associated with advanced atherosclerosis (chronic periaortitis).

AIMS: To determine whether aortic adventitial chronic inflammation associated with advanced atherosclerosis ("chronic periaortitis") is associated with any detectable cytokine gene expression. METHODS: RNA was extracted from six fresh surgical specimens of atherosclertic aortic aneurysm wall showing a spectrum of chronic periaortitis. Controls included four normal aortas and an HUT 78 T cell line. Reverse transcriptase and the polymerase chain reaction (PCR) were used to amplify mRNA for interleukins-1 alpha (IL-1 alpha), -2 (IL-2), -4 (IL-4), IL-2 receptor-alpha (IL-2R-alpha), tumour necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) with beta-actin as an internal control. RESULTS: No TNF-alpha mRNA was detected in any of the inflamed aortic tissue samples, in contrast to the aortic T lymphocytes propagated in culture in IL-2 conditioned medium (aortic cultured T cells) and peripheral blood mononuclear cells from these patients. In contrast, IFN-gamma, IL-1 alpha, IL-2, IL-2 receptor and IL-4 PCR products were detected for each inflamed aortic tissue RNA sample with IFN-gamma mRNA expression increasing with increasing degrees of adventitial inflammation. Only beta-actin mRNA was present in the normal aorta. CONCLUSIONS: These findings indicate the active nature of aortic adventitial chronic inflammation associated with human advanced atherosclerosis ("chronic periaortitis") and show its possible progressive potential to the clinically important diseases termed "idiopathic retroperitoneal fibrosis" and "inflammatory aneurysm".

The distribution of adhesion molecules in chronic periaortitis.

Chronic periaortitis is a local complication of human atherosclerosis. It is defined as the triad of advanced atherosclerosis, medical thinning and aortic adventitial chronic inflammation. It is present to a variable degree in association with atherosclerotic abdominal aortic aneurysms. These aortic adventitial infiltrates differ from those described solely within the atheroma itself, in that they consist predominantly of B lymphocytes. Many of the lymphocytes are activated and proliferating, and germinal centres are common. In this study, an immunohistochemical analysis was carried out on fresh surgical aortic aneurysm tissue in order to investigate the presence and distribution of activation-inducible adhesion molecules, and to correlate this with the degree of inflammation. A consistent finding was the presence of E-selectin on endothelial cells in up to 50% of the vessels throughout the aortic wall and at the base of the atheroma, independent of the severity of inflammation. ICAM-1 expression was abundant on many cell types and increased with the severity of chronic inflammation, being strongest in the germinal centres. VCAM-1 expression was predominant on follicular dendritic cells and also increased with severity of inflammation. VCAM-1 expression was also detected on vessels within lymphoid follicles. The pattern of expression of the adhesion molecules suggests a role in the initiation and progression of chronic inflammation associated with advanced atherosclerosis.

Quantification of specific mRNA by flatbed scintillation counting of dual-labeled dot blots.

A dual-labeling technique was developed for direct quantification of specific mRNA using a flatbed liquid scintillation counter. This method simultaneously measures cpm of 32P- and 35S-labeled probes bound to RNA dot blots and subtracts counts due to nonspecific background radioactivity bound to the filter. Probes for T-cell receptor and beta-actin (as the internal standard) were hybridized both separately and simultaneously to RNA isolated from five different sources. There was concordance between the radioactivity measured from single- and dual-hybridizations for each combination of 35S- and 32P-labeled probes. This methodology directly quantifies specific mRNA sequences bound to membranes and has potential for measuring gene dosage, without the need for re-probing or densitometric analysis.

Papillomavirus screening in cervical cell samples using dual-label dot-blot analysis.

The presence of human papillomavirus (HPV) in cervical cells is closely related to the development of cervical carcinoma. Detection of virus may be by Southern blot, dot blot or the highly sensitive polymerase chain reaction. Whatever method is employed, there are problems of false negatives due to poor clinical samples in which the DNA may be degraded or is absent altogether. Here we describe a new method of dual labelling for dot blots using a 32P-labelled probe for HPV and a 35S-labelled probe for human actin genes. The samples were counted on a Beta-plate flat-bed scintillation counter and the data analysed to separate the activities of the two isotopes. The counts from the actin probe show whether human DNA is present or not and false negatives from this cause may thereby be eliminated. The counts due to HPV when compared with those for actin give a quantitative measure of HPV abundance for the particular sample and this may have clinical relevance.

The distribution of adhesion molecules in human atherosclerosis.

Chronic inflammatory cells are a recognized component of atherosclerotic plaques at all stages of development. As adhesion molecules play a fundamental role in inflammatory processes, we have carried out an immunohistochemical investigation of the distribution of endothelial leucocyte adhesion molecule-1 (ELAM-1)*, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human atherosclerotic lesions. Autopsy specimens from abdominal aorta and coronary arteries were obtained from 21 cases within 24 h of death. ELAM-1 and ICAM-1 were consistently expressed by the entire intimal endothelium of normal coronary arteries and also by the intimal endothelium overlying aortic fatty streaks. Both coronary artery and aortic lesions showed strong staining for ICAM-1 on and around macrophages. VCAM-1 was not detected on intimal endothelial cells, but strong staining of adventitial lymphoid aggregates for this molecule was seen. This work suggests a role for ELAM-1 and ICAM-1 in mononuclear cell recruitment during atherogenesis.

Quantification of oncogene dosage in tumours by simultaneous dual-label hybridization.

Gene amplification and allele loss occur in a variety of human tumours and some have prognostic value. Therefore, techniques which facilitate detection and quantification of gene dosage could have wide applicability in cancer research. Using the INT-2 gene as a model system, a quantitative procedure has been developed for measuring gene copy number using dual-label hybridization to DNA dot blots. A probe specific for the INT-2 gene was labelled with [alpha-32P]dCTP and a probe to beta-actin, the control locus, was labelled with [alpha-35S]dATP. Flat-bed scintillation counting was used to detect and separate the emissions resulting from each bound probe, and gene dosage was calculated from the ratio of INT-2 to the beta-actin probe compared with the ratio derived from constitutional DNA. Calculated ratios of greater than 1.22 and less than 0.78 indicated gene amplification and allelic loss respectively, at the 99% confidence limit derived from the population of 35 constitutional DNAs. The results were validated by RFLP analysis. It is expected that this technique will permit precise gene dosage quantification in many areas.

Combined immunohistochemical and immunofluorescence method to determine the phenotype of proliferating cell populations.

AIMS: To determine the phenotype of proliferating cell populations. METHODS: The double immunostaining technique combines the autofluorescent properties of alkaline phosphatase substrate naphthol/Fast Red with immunofluorescence using fluorescein. Fresh human tonsil and fresh atherosclerotic aortic aneurysm wall tissue were studied using a panel of monoclonal antibodies including Ki-67, CD4, CD8, CD19, CD22, HLA-DR alpha, CD68 and CD31. RESULTS: This double immunostaining method permitted simultaneous colocalisation of different markers on the same cell and could be used to identify HLA-DR positive cells as well as proliferation associated Ki-67 positive cells in human tonsil tissue and in chronic periaortitis associated with advanced atherosclerosis. CONCLUSION: This technique is simple and the results may be viewed using a single fluorescence filter. The Fast Red reaction product is stable and does not fade under storage. The staining works particularly well with markers for nuclear antigens in combination with markers for cytoplasmic or surface antigens.

Basic fibroblast growth factor and interleukins 4 and 6 stimulate the release of IFN-gamma by individual NK cells.

Both the secretory and cytotoxic activity of natural killer (NK) cells are known to be regulated by such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In the present study we have used the reverse hemolytic plaque assay to investigate either the direct effects of the protein kinase activator, phorbol myristate acetate (PMA), or exposure to recombinant human interleukins 2, 4, and 6 (IL-2, IL-4, and IL-6) tumour necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) on the release of IFN-gamma by individual, immunoidentified NK cells isolated from peripheral blood. This sensitive immunoassay was adapted and coupled with immunocytochemistry not only to immunophenotype and enumerate cells secreting IFN-gamma in a given cell population, but also to quantify the amount of this cytokine released per individual cell. These studies have confirmed mononuclear cells with the morphology of large granular lymphocytes and the immunophenotype of CD3-/CD16+ NK cells to be the predominant source of spontaneously released IFN-gamma in vitro. In contrast to this, fewer than 2% of the CD3+ T cells secreted detectable levels of this cytokine during the assay, irrespective of the stimulus applied. Whilst TNF-alpha had no significant effect on IFN-gamma release by NK cells, a 6-hr exposure to IL-2 or PMA stimulated an increase in the amount secreted per single cell. Furthermore, bFGF and interleukins 4 and 6 elicited a marked, dose-dependent stimulation of IFN-gamma secretion by this cell type. However, exposure to these cytokines did not alter the number of cells capable of releasing detectable levels of IFN-gamma during the assay. These studies demonstrate that (i) both the spontaneous and stimulated release of IFN-gamma by NK cells can be visualized and quantified at the single-cell level using this sensitive immunoassay, and (ii) bFGF and interleukins 2, 4, and 6, but not TNF-alpha, are potent stimulants of IFN-gamma secretion by CD3-/CD16+ NK cells.

Immunohistochemical characterization of inflammatory cells associated with advanced atherosclerosis.

During repair of 12 atherosclerotic abdominal aortic aneurysms, fresh samples of aneurysm wall were obtained. Histology confirmed the presence of advanced atherosclerosis associated with medial thinning and a variable aortic adventitial chronic inflammatory cell infiltrate. Monoclonal antibodies were used to identify the inflammatory cells throughout the aortic wall. The majority of lymphocytes in the aortic adventitia were B-cells. B-cells were not present in atheromatous plaques. T-cells, predominantly T-helper cells, were found in atheromatous plaques and in aortic adventitia. The majority of lymphocytes and macrophages in aortic adventitia and most vascular endothelial cells were HLA-DR positive. Ki-67 staining was found in B-cells and T-helper cells, indicating that these cells were proliferating. Occasional lymphocytes were BerH2 positive, indicating that some lymphocytes were activated. These findings suggest that chronic periaortitis is an active, immunologically mediated, local complication of advanced human atherosclerosis.