As a result of a peritoneal inflammation, mononuclear phagocytes expressing only few receptors for α2-macroglobulin-protease complexes are recruited. About 10 h after the initiation of the inflammation, the resident macrophages possessing a higher concentration of receptors than the newly recruited cells, and later the recruited monocytes, up-regulate the concentration of receptors for α2-macroglobulin-protease complexes as measured by FACS analysis of receptor-bound FITC-conjugated α2-macroglobulin-trypsin complexes.

Human factor VIII (procoagulant: VIII:C, coagulant antigen: VIII:Ag) is a hetero-dimeric plasma protein consisting of 80 kDa and 90–120 kDa polypeptides linked by divalent metal-ion bridges. In plasma F. VIII circulates non-covalently bound to von Willebrand factor. In spite of recent advances in biochemistry of F. VIII, the specific cellular origin of this protein is still obscure. In the past, several animal studies have failed to clearly indicate the organ most important for F. VIII production (Review 1). Recently it was shown that liver transplantation to a haemophilia A patient resulted in normalization of plasma VIII:C [2]. The molecular cloning of human factor VIII was performed using A DNA library from human liver [3]. Two attempts to demonstrate VIII: Ag in liver tissue biopsies gave inconsistent results, although the same monoclonal antibody was used. In the first study [4] VIII: Ag immune reactivity was disclosed to liver sinusoideal lining cells, whereas the second study [5] demonstrated VIII: Ag in endoplasmatic reticulum of hepatocytes, the site of protein synthesis, but some VIII:Ag reactivity was also seen in sinusoideal cells. It therefore is reasonably assumed that F. VIII synthesis takes place in the liver. The present investigation, performed on isolated and cultivated human hepatocytes, indicates that the hepatocyte is a production site for F. VIII.

125I-labelled alpha 2-macroglobulin-trypsin (alpha 2MT) complex bound specifically to freshly isolated human blood monocytes at 4 degrees C but not to B or T lymphocytes, polymorphonuclear leucocytes, erythrocytes or thrombocytes. Binding of 12 pmol/l labelled alpha 2MT to freshly isolated monocytes was low. However, when monocytes were cultured in vitro, binding increased, reaching 10- to 20-fold higher specific binding by 2-4 weeks. Non-specific binding was absent. Considerable variations were observed in binding to monocytes from different cultures (individuals). The half-time of 125I-labelled alpha 2MT complex association to 14-days-old monocyte cultures was about 6 h at 4 degrees C. Dissociation of labelled complex after the addition of a saturing concentration of unlabelled complex was biphasic. About half of the labelled complex dissociated with a half-time of about 1 h, whereas the other half dissociated extremely slowly. At near steady state, half of the receptors were occupied at a complex concentration of about 200 pmol/l and Scatchard analysis showed that the data were adequately described by assuming one class of receptors. It is concluded that human monocytes express a marked increase in binding of alpha 2M complex when developing to macrophage-like cells in tissue cultures.

Human T‐lymphocyte clones specific for antigenic components of purified protein derivative (PPD) of tuberculin were generated by limiting dilution using in vilro PPD‐activated peripheral blood mononuclear cells from a single donor. The HLA restriction specificity of eight clones that were cytotoxic against autologous PPD‐pulsed monocyte targets, was examined against a panel of allogeneic PPD pulsed targets, In agreement with our findings with bulk‐expanded PPD‐reactive cytotoxic T lymphocytes, all clones were restricted by HLA class II antigens: seven by HLA‐DR 2 and one by HLA‐DRwlO‐the other HLA‐DR antigen of the donor. All clones were CD3+, CD44, CD8‐. One clone exhibited, in addition to HLA‐DR2 restriction, unrestricted cytotoxic alloreactivity against HLA‐DR1. In monoclonal antibody‐blocking experiments the latter clone was the only one that was blocked. Its lytic ability was abolished by two monoclonal antibodies against monomorphie HLA DR determinants. The antigen specificity of the clones was studied by using autologous monocyte targets pulsed with antigens prepared from a range of different mycobacterial species. All seven HLA‐DR2‐restricted clones reacted with the majority of antigens tested. In contrast, the HLA‐DRwlO‐restricted clone reacted exclusively with an antigen unique to PPD.

A simple, sensitive and precise enzyme-linked immunosorbent assay for the quantitation of alpha 2-macroglobulin (alpha 2M) in supernatants of cell cultures was constructed. All reagents apart from the alpha 2M standard were commercially available. The assay range was 2.0-500 micrograms/l. The intra-assay coefficient of variation (CV%) was 4.6%, and the imprecision between runs was 8.9% at 10 micrograms/l and 9.0% at 110 micrograms/l. Recovery of alpha 2M, added to cell culture medium free of serum, was 97.5 +/- 7.2% (mean +/- SD) and the recovery of alpha 2M added to pooled human serum was 101 +/- 6.0%. There was no significant difference between the recovery of alpha 2M-standard and alpha 2M-trypsin complexes, whereas the dose response of a commercial alpha 2M-standard was lower than expected (81.1 +/- 7.5%), indicating a lower purity and/or conformational changes in the epitopes of this reagent. As expected, supernatants of mononuclear lymphocyte cultures enriched in monocytes contained significantly higher concentrations of alpha 2M than supernatants of cell cultures depleted in monocytes. Our results indicate that the ELISA method could be useful tool in the study of the alpha 2M turnover in all cell cultures in vitro.