Characteristics of human synovial fibroblast activation by IL-1 beta and TNF alpha.

Human synovial fibroblast cell lines (HSN), established from tissues obtained from the knee joints of arthritis patients undergoing arthoplasty, were used to investigate the effects of human interleukin-1 (IL-1) beta and tumour necrosis factor (TNF)alpha on proliferation and prostaglandin E2 (PGE2) secretion. IL-1 beta and TNF alpha were equipotent stimulators of HSN proliferation. Classical non-steroidal anti-inflammatory drugs and glucocorticoids significantly augmented this effect. In addition, IL-1 beta and TNF alpha were potent inducers of PGE2 production while exogenous PGE2 was growth inhibiting. These data suggest that the secretion of PGE2 by monokine-stimulated HSN exerts a negative feedback signal. Further examination of IL-1 beta- and TNF alpha-induced PGE2 secretion revealed IL-1 beta to be a more potent stimulator; however, this observation may be due, in part, to differences in the kinetics of induction. Rabbit anti-IL-1 beta and anti-TNF alpha specifically neutralized both proliferation and PGE2 production induced by these monokines, but anti-IL-1 beta (or anti-IL-1 alpha) did not block TNF alpha activity. It is unclear whether TNF alpha stimulates HSN to produce IL-1, but the antibody data suggest that extracellular IL-1 is not responsible for TNF alpha in vitro activity.

7C3, a marker for the differentiation of human macrophage cell lines.

The induction of differentiation in the human monoblastlike cell line U937 by 1,25 dihydroxyvitamin D3, interferon gamma, or phorbol esters is associated with the expression of a novel surface antigen, 7C3. The expression of 7C3 is maximal after 24 hr and is dependent upon de novo protein synthesis. The appearance of 7C3 during U937 differentiation is inhibited by dexamethasone while the increased expression of the macrophage marker OKM1 is not affected. 7C3 antigen is also expressed on HL60 cells when induced along the macrophage pathway and is expressed weakly on peripheral blood monocytes but not on lymphoid cells or granulocytes. 7C3 expression is sensitive to trypsin and low concentrations of the nonionic detergents NP-40 and Triton X-100. The induction of 7C3 expression may serve as a useful model to understand the regulatory events involved during the early phases of monocyte-macrophage differentiation.

In vivo expression and regulation of murine IL 2 receptors after antigen sensitization.

We have studied the in vivo regulation and expression of the murine IL 2 receptor after antigen sensitization. High and low affinity receptor expression has been studied by flow cytometry analysis and radiolabeled IL 2 binding. Both anti-IL 2 receptor antibody and unlabeled IL 2 inhibited the radiolabeled IL 2 binding. The kinetics of expression of the IL 2 receptor closely correspond to the proliferative response, as assessed by IUdR radioisotope uptake. The expression of IL 2 receptors correlated with the proliferative response profile of the murine strains surveyed during a study of responses to picryl chloride. Neither immune compromised 4-mo-old MRL/lpr mice nor athymic nude mice generated significant antigen-specific proliferative responses or IL 2 receptor expression after antigen sensitization. Mice rendered specifically unresponsive to antigen displayed reduced proliferative responses to the tolerizing antigen, as well as reduced numbers of IL 2 receptor-positive cells. Finally, the treatment of mice with immunosuppressive drugs reduced both the proliferative responses as well as the number of IL 2 receptor-positive cells. However, at the cellular level, the drugs produced different effects on IL 2 receptor expression.

Characterization and specificity of a high titre polyclonal goat anti-murine IL-1 antibody.

The immunization of goats with an HPLC-purified IL-1 preparation derived from the supernatants of LPS-induced P388D1 cultures has resulted in the derivation of a high titre antiserum specific for murine IL-1. This antiserum exhibits a 50% inhibition of an IL-1-dependent thymocyte costimulation assay at a reciprocal dilution of 30,000 and antibody activity is still detected at 100,000-fold dilution. The goat anti-murine IL-1 serum is specific for murine IL-1 synthesized by several murine macrophage cell lines and does not neutralize human, rabbit or porcine (catabolin) IL-1. The antiserum also inhibits antigen-induced proliferation of the D10.G4 helper T-cell line. In addition to the reaction against IL-1, the antiserum also detects three additional peptides with molecular weights between 20,000 and 30,000.

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum.

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Cell sorting using a universally applicable affinity chromatography matrix: solid-phase anti-fluorescein isothiocyanate antibody.

Procedures are described for fractionating cells utilizing a universally applicable cellular affinity chromatography matrix. The affinity matrix consists of immunoabsorption purified goat anti-fluorescein isothiocyanate antibody coupled to large derivatized polyacrylamide beads. This matrix may, in principle, be used to isolate any cell subpopulation provided it has a fluorescein-labeled ligand on its surface. In this report the matrix was used to isolate viable purified fractions of mouse surface Ig-positive cells, Lyt1 cells, and mouse lymphocytes that bind the lectin soybean agglutinin. A preliminary experiment using the anti-FITC beads suggested that this technique can provide a fraction of cells enriched in antigen binding cells. Cell populations isolated by this technique retain their ability to respond to in vitro mitogen stimulation, as well as their ability to be maintained in cell culture following fractionation. Additional experiments using a column consisting of goat anti-rabbit Ig antibody coupled to the same support material are also reported.

Isolation of three separate anaphylatoxins from complement-activated human serum.

Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a. The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.

A sensitive and reproducible assay for the quantitation of erythrophagocytosis and its correlation with reduction in tritiated thymidine uptake in a tumor target cell system modified by immunoenhancing or immunosuppressive agents.

A new and sensitive assay procedure for studying erythrophagocytosis is described. The assay technique permits quantitation of the in vivo and in vitro effects of chemicals, hormones, and cell, or microbrial products, on the level of phagocytic activation of glass-adherent cells. The effect of intraperitoneal injection of BCG, Zymosan, Vitamin A, B. pertussis, cortisone, estrone, and thioglycollate on phagocytic activation of peritoneal exudate cells harvested from two days up to 28 days following drug injection was examined by this assay. Erythrophagocytosis was compared to the effect of "activated" spleen cells on tritiated thymidine uptake of a tumor target cell suspension.