TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.

Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing.

Tumour necrosis factor (tumour necrosis factor-alpha/cachectin) plays a critical role in certain physiological defensive responses but causes severe damage to the host organism when produced in excess. There are two forms of tumour necrosis factor, a type II membrane protein of relative molecular mass 26,000 (26K) and a soluble, 17K form generated from the cell-bound protein by proteolytic cleavage. The two forms of tumour necrosis factor and lymphotoxin-alpha (tumour necrosis factor-beta/lymphotoxin), a related protein, have similar but apparently not identical biological activities. A therapeutic agent which inhibited the release of tumour necrosis factor, but did not reduce the cell-associated activity or the level of lymphotoxin-alpha, might preserve the benefits of these cytokines while preventing tumour necrosis factor-induced damage. Here we describe a potent inhibitor of tumour necrosis factor processing and report that it protects mice from a lethal dose of endotoxin.

Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists.

Two forms (monomeric or dimeric) of the extracellular, ligand-binding portion of the human p80 cell-surface receptor for TNF were used to antagonize TNF activity in vitro and in vivo. The dimeric sTNFR:Fc molecule was a more potent inhibitor of TNF than the monomeric sTNFR (50 to 1000x), as assessed in vitro by inhibition of TNF binding or bioactivity and in vivo by protection of mice from an otherwise lethal injection of LPS. Surprisingly, the dimeric sTNFR:Fc construct demonstrated a beneficial effect even when administered 3 h after a lethal LPS injection (i.e., after serum TNF levels had peaked and receded). To study the mechanism by which the soluble TNFR functions in vivo, serum TNF levels were examined in mice given LPS in the presence or absence of soluble receptor. Administration of a mortality-reducing dose of sTNFR:Fc ablated the rise in serum TNF bioactivity that normally occurs in response to LPS. However, TNF bioactivity was revealed in these "TNF-negative" serum samples when the L929 bioassay was modified by inclusion of a mAb that blocks the binding of murine TNF to the human soluble TNFReceptor. These results indicate that the absence of direct cytolytic activity in the L929 assay was caused by neutralization of TNF, rather than to an absence of TNF in the serum. Moreover, administration of either monomeric sTNFR or low doses of dimeric sTNFR:Fc actually resulted in increased serum TNF levels compared to mice given LPS but no soluble receptor. However, these "agonistic" doses of soluble receptor did not lead to increased mortality when an LD60 dose of LPS was given. Thus, dimeric sTNFR are effective inhibitors of TNF and under some circumstances function simultaneously as both TNF "carriers" and antagonists of TNF biologic activity.

Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery.

Experimental autoimmune encephalomyelitis (EAE) serves as an important animal model for understanding the events that lead to immune-mediated inflammation and tissue destruction within the central nervous system. We have utilized a murine adoptive transfer model of EAE and semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine cytokine mRNA expression within the central nervous system in relation to the onset and resolution of paralysis associated with EAE. Spinal cord samples, obtained from mice as they progressed through discrete clinical stages of EAE, were examined for the expression of six cytokine genes (IL-1 alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma). Distinct patterns of cytokine gene expression were observed during the acute, recovery, and chronic phases of EAE. The acute phase of disease was characterized by rapid increases in the levels of mRNA for IL-2, IL-4, IL-6, IFN-gamma, and IL-1 alpha. In fact, peak expression of several cytokine mRNA (e.g., IL-2, IL-4, IL-6, and IFN-gamma) occurred before the peak in clinical severity. In contrast, IL-1 alpha mRNA levels were elevated throughout the initial disease course. IL-10 mRNA demonstrated only modest increases during the acute phase of EAE. Stabilization of the clinical symptoms was characterized by rapid declines in the mRNA levels of IL-2, IL-4, IL-6, and IFN-gamma. The decreases in these four cytokine mRNA levels occurred concomitant with a dramatic rise in IL-10 mRNA. Finally, of the six cytokine mRNA examined, only IL-1 alpha, IFN-gamma, and IL-10 mRNA remained elevated during the early chronic stage. These results suggest that local cytokine production varies significantly during the course of EAE and that increases in discrete sets of cytokines are associated with the acute response and the recovery/chronic phase of disease.