Urine of some febrile patients exhibits a TNF-alpha inhibitory activity (TNF-alpha INH), sensitive to heat and trypsin, with an apparent mol wt of 40-60 X 10(3) and a pI range of 5.5-6.1. As for the Il-1 INH, the TNF INH activity involves a competitive mechanism of action suggesting the existence of a family of negative feedback-regulating molecules interfering with cytokines actions.

Interleukin 1 (IL-1) possesses multiple biological activities that may be blocked selectively by different inhibitors. Some known inhibitors block the lymphocyte activating factor (LAF/IL-1) but not the mononuclear cell factor (MCF/IL-1) measured by its capacity to stimulate prostaglandin E2 (PGE2) and colingenase production. The presence of IL-1 in vivo may be difficult to detect due to the presence of inhibitor(s) and the level of the inhibitor(s) may vary depending upon pathological conditions. We have found that urine from three patients with monocytic leukemia (M5) contained high levels of inhibitor(s) of MCF/II-1, whereas urine of normal subjects did not contain significant amounts. Urine from two patients with other blood neoplasic diseases also contained little inhibitory activity. The MCF/IL-1 inhibitor(s), which also acts on human recombinant IL-1 ft, is -25-35 kD, is not retained on concanavalin A-Sepharose column and can be partially destroyed with urea and boiling. At this stage of the purification the fraction containing the MCF/IL-1 inhibitor(s) also inhibits the LAF/IL-1 assay. However, this inhibitor(s) is probably distinct from other inhibitors already described.

Previous studies have shown that urine of febrile patients contains a tumor necrosis factor a inhibiting activity (TNF-a Inh) when tested in a cytotoxicity assay using the tumor necrosis factor a (TNF-a)-susceptible cell line L929. In the present study, we investigated the relationship between the TNF-a Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-a activities by binding to the ligand. We demonstrate that human TNF-et is affected to a greater extent than is murine TNF-a. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-a Inh that neutralizes its activity and does not recognize TNF-a. Solubilized cross-linked '25I-labeled TNF-a receptor complex could be immunoprecipitated by using either anti-TNF-a or anti-TNF-a Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-a, it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-a receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-a Inh antibody revealed with a fluorescein isothiocyanatecoupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-a Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-at Inh originally described might be a soluble form of the TNF receptor itself.

Guinamard R, Fougereau M, Seckinger P. The SH3 Domain of Bruton's Tyrosine Kinase Interacts with Vav, Sam68 and EWS. Scand J Immunol 1997; 45: 587-595 Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro → Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.

The urine of patients with fever above 39 degrees C contains an inhibitor of interleukin 1. Herein we describe the purification of the interleukin 1 inhibitor by ion-exchange chromatography, hydrophobic chromatography, gel filtration and negative immunosorption. The purified protein has a molecular weight of 26,000; its size is reduced to 24,000 upon treatment with endoglycosidase F. The IL 1 inhibitor is a competitive inhibitor and acts by binding to the IL 1 receptor. The inhibitor recognizes the murine and human IL 1 receptor with similar affinity. The purified IL 1 inhibitor has a specific activity of 3 x 10(7) to 4.5 x 10(7) units/mg as tested on the human astrocytoma (U-373) cell proliferation bioassay and murine EL4.6.1 cell IL 1 receptor-binding assay.

Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.

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