InVivoMAb anti-mouse TNFR1 (CD120a)

Monoclonal antibodies (mAbs) specific for the murine p55 and p75 tumor necrosis factor (TNF) receptors were produced after immunization of Armenian hamsters with the purified soluble extracellular domains of each receptor protein. Four p55- (55R) and five p75 (TR75)-reactive mAbs immunoprecipitated the appropriate receptor from the surface of L929 cells. None of the mAbs cross-reacted with the other TNF receptor form. The mAbs were functionally characterized by their ability to inhibit ligand binding and influence TNF-dependent L cell cytolytic activity or proliferation of the murine cytolytic T cell clone CT6. One p55-specific mAb, 55R-593, displayed agonist activity, while two other p55-specific mAbs (55R-170 and -176) were found to be TNF antagonists. The fourth mAb (55R-286) had no functional effects on cells. Several antibodies specific for the p75 TNF receptor partially inhibited recombinant murine TNF-alpha-dependent cytolytic activity (60%). Blocking mAbs specific for p75 but not anti-p55 inhibited TNF-mediated proliferation of CT6 T cells. When used in vivo, p55- but not p75-specific mAbs protected mice from lethal endotoxin shock and blocked development of a protective response against Listeria monocytogenes infection. In contrast, both p55 and p75 mAbs individually blocked development of skin necrosis in mice treated with murine TNF-alpha. These data thus demonstrate the utility of the two families of murine TNF receptor-specific mAbs and identify a novel function of the p75 TNF receptor in vivo.

The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of "ligand passing," whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using the in vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit.

The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of "ligand passing," whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using the in vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit.

In spontaneous inflammatory arthritis of K/BxN T cell receptor transgenic mice, the effector phase of the disease is provoked by binding of immunoglobulins (Igs) to joint surfaces. Inflammatory cytokines are known to be involved in human inflammatory arthritis, in particular rheumatoid arthritis, although, overall, the pathogenetic mechanisms of the human affliction remain unclear. To explore the analogy between the K/BxN model and human patients, we assessed the role and relative importance of inflammatory cytokines in K/BxN joint inflammation by transferring arthritogenic serum into a panel of genetically deficient recipients. Interleukin (IL)-1 proved absolutely necessary. Tumor necrosis factor (TNF)-alpha was also required, although seemingly less critically than IL-1, because a proportion of TNF-alpha-deficient mice developed robust disease. There was no evidence for an important role for IL-6. Bone destruction and reconstruction were also examined. We found that all mice with strong inflammation exhibited the bone erosion and reconstruction phenomena typical of K/BxN arthritis, with no evidence of any particular requirement for TNFalpha for bone destruction. The variability in the requirement for TNF-alpha, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.