A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Reversal of virus-induced systemic shock and respiratory failure by blockade of the lymphotoxin pathway.

At present, little is known about the pathogenesis of acute virus-induced shock and pulmonary failure. A chief impediment in understanding the underlying disease mechanisms and developing treatment strategies has been the lack of a suitable animal model. This study describes a mouse model of virus-induced systemic shock and respiratory distress, and shows that blockade of the lymphotoxin beta receptor pathway reverses the disease.

Fourth component of human complement: description of a three polypeptide chain structure.

The fourth component of human complement (C4) was shown to be composed of three distinct polypeptide chains linked by disulfide bonds and noncovalent forces. The sum of the molecular weights of the chains equalled that of the intact molecule. The mol wt of the alpha-, beta-, and gamma-chains were respectively, 93,000, 78,000, and 33,000 daltons. Action of C1s on C4 affected only the alpha-chain, reducing its mol wt to 87,000 daltons. The size of the activation peptide. C4a, is therefore estimated to be 6,000 and that of the major fragment C4b, 198,000 daltons. Periodic acid-Schiff-stained SDS polyacrylamide gels of reduced C4 revealed carbohydrate to be associated with all three chains. A modification of the original method of isolation of C4 is presented.

Identification of a T cell hybridoma that produces large quantities of macrophage-activating factor.

A murine T cell hybridoma, constructed by fusion of alloantigen-activated T cells with the BW5147 T cell lymphoma, which produces a lymphokine capable of inducing tumoricidal activity in macrophages, has been identified. Lymphokine release could be detected only after mitogen stimulation of the T cell hybridoma culture. Upon cloning of the parental hybridoma, 24 out of 27 clones produced tumoricidal-inducing activity. Seven clones produced more cytocidal-inducing activity than did conventional supernatants, generated by concanavalin A stimulation of normal murine spleen cell cultures, which contained macrophage-activating factor (MAF). The supernatant of hybridoma clone 24/G1 was 25 times more active than conventional MAF preparations. Using supernatants from a variety of clones, the levels of macrophage-activating activity and interleukin 2 were found to vary independently of one another. The lymphokine produced by hybridoma clone 24/G1 appeared to be identical to conventional MAF by a variety of criteria including: (a) a requirement for a second signal for induction of tumoricidal activity in macrophages, (b) inactivation after incubation for 1 h at 65 degrees C, and (c) loss of activity after treatment at pH 4.0 but not at pH 5.0. Like conventional MAF, the hybridoma MAF eluted as a single peak after molecular sieve chromatography on Sephadex G100 and exhibited an apparent molecular weight of 55,000. Although somewhat heterogeneous, the majority of hybridoma 24/G1 MAF displayed an isoelectric point of 5.4 as determined using the chromatofocusing technique. These results thus illustrate the usefulness of T cell hybridomas in distinguishing between various lymphokine activities and indicate that the T cell hybridoma clone 24/G1 will be of particular usefulness in achieving the biochemical purification of substantial quantities of murine MAF.

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.

Gamma interferon limits access of Listeria monocytogenes to the macrophage cytoplasm.

The effect of rIFN-gamma and rTNF on the fate of hemolytic and nonhemolytic (hly-) Listeria monocytogenes in cultured mouse peritoneal macrophages was investigated. In untreated macrophages, approximately 80% of the hemolytic bacteria were killed during the first 2 h of incubation, but the survivors doubled between two and three times. In rIFN-gamma-treated macrophages, although the bacterial killing was identical to the controls during the first 2 h, there was no subsequent bacterial growth, and bactericidal activity continued for the duration of the experiment. rTNF has no affect by itself, but acted synergistically with rIFN-gamma to promote bacterial killing. Infected macrophages with or without rIFN-gamma were examined by EM. The results clearly showed that the role of rIFN-gamma was to prevent access of L. monocytogenes to the macrophage cytoplasm, which would prevent cell-to-cell spread of the bacteria. In addition, rIFN-gamma-treated macrophages exhibited enhanced digestive capacity of the intracellular bacteria.

Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor.

Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.

Alternative pathway of complement: demonstration and characterization of initiating factor and its properdin-independent function.

A novel component of the properdin system has been discribed which represents a heretofore unrecognized human serum protein. The protein has been tentatively termed the initiating factor (IF) because it functions in the initial reaction of the properdin pathway. IF is a 170,000 dalton beta-pseudoglobulin which is composed of two presumably identical 85,000 dalton chains linked by disulfide bonds. The protein reacts with antibody to nephritic factor, which is defined by its activity and is found in the serum of patients with certain nephritides. The activity of IF is heat stable. Upon treatment of serum with activators of the alternative pathway, the initial C3 convertase is assembled from IF, Factors D and C, C3, and magnesium without participation of properdin. It is the function of the enzyme to deposit C3b on the surface of the activator particles, thereby affording generation of the solid phase enzymes of the pathway, a process that is a prerequisite for properdin activation. By exposure to low pH, IF assumed the electrophoretic mobility of psi-globulin and acquired the ability to generate without activators a fluid phase C3 convertase in serum. Serum depleted of IF did not allow activation of the properdin pathway. Serum depleted of properdin did permit activation of the pathway and expression of cytolytic activity. These results raise the possibility that IF represents the recognition unit of the pathway.

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity.

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.

Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.

Activation of the alternative pathway of complement commences with the formation of an initial fluid-phase C3 convertase. Treatment of C3 with the nucleophilic reagent methylamine has previously been shown to result in the cleavage of an intramolecular thioester bond and to induce C3b-like properties, including the ability to form a fluid-phase C3 convertase. This report examines the hypothesis that spontaneous hydrolysis of the thioester generates a derivative of C3 that is responsible for the formation of the initial C3 convertase of the alternative pathway. The rate of spontaneous decay of C3 hemolytic activity in buffer was found to be between 0.2 and 0.4%/h. In the presence of other alternative pathway proteins, the rate of inactivation was 1%/h. The rate of spontaneous inactivation was greatly accelerated by low concentrations of chaotrophic agents such as KSCN or guanidine. Liberation of a sulfhydryl group, not present in native C3, correlated with loss of hemolytic activity, indicating that exposure to chaotropic agents resulted in thioester hydrolysis. Unlike native C3, C3 bearing a single reactive sulfhydryl group was capable of generating fluid-phase C3 convertase with Factors B, D, and P and was cleaved by Factor I (C3b inactivator) in the presence of Factor H (beta 1H). The fragmentation patterns indicated that the C3a domain was covalently associated with the functionally C3b-like C3. Organomercurial agarose was employed for the rapid removal of sulfhydryl-bearing, hemolytically inactive forms of C3 and C3b from native hemolytically active C3.

Interaction of target cell-bound C3bi and C3d with human lymphocyte receptors. Enhancement of antibody-mediated cellular cytotoxicity.

The occurrence and distribution of distinct receptors for three C3 fragments on purified human blood lymphocytes were studied by rosette formation. Indicator cells were bovine, chicken, or sheep erythrocytes (E) bearing up to 100,000 molecules of human C3b (EC3b) without antibody. EC3b was converted to C3bi-bearing-E (EC3bi) with purified C3b inactivator (factor I) and beta1H (factor H), and to C3d-bearing E (EC3d) by treatment of EC3bi with trypsin. Using bovine E (Eb) as indicators, approximately 11% of the lymphocytes bound EbC3b, 6% bound EbC3bi and 2% bound EbC3d. Fractionation of the lymphocytes by adsorption to monolayers of C3-fragment-bearing Eb or by rosetting indicated that most of the cells with receptors for C3b were distinct from those having receptors for C3bi and/or C3d. Cells from two lymphoblastoid cell lines (Raji and Daudi) formed strong rosettes with EC3b, which were weak. 51Cr-labeled E was used as a target in antibody, C3-fragment-bearing E was not lysed by the lymphocytes. However, at suboptimal concentrations of IgG enhancing capacity of the fragments occurred in the order of C3bi greater than C3d greater than C3b. In addition, C3-fragment-bearing cells inhibited the lysis of antibody-coated cells not concluded that target cell bound C3 fragments enhance ADCC by improving contact between target cells and those effector cells which have C3 receptors. Cell-bound C3 effector cells. It is proposed that certain lymphocytes are capable of interacting with C3bi in addition to C3b and C3d and that C3bi and C3d have a greater regulatory effect on their cytolytic function than C3b.

Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

The complement regulatory enzyme, C3b inactivator (C3bINA), has been purified from human serum by affinity chromatography on an anti-C3bINA Sepharose column. Subsequent chromatography on DEAE-cellulose and removal of IgG with anti-IgG Sepharose resulted in a product which was found to be homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecule is composed of two disulfide bonded polypeptide chains with mol wt of 50,000 and 38,000 daltons. Human CobINA was found to be a glycoprotein containing at least 10.7% carbohydrate and to have a normal serum concentration of 34 +/- 7 mug/ml (mean +/- 1 SD). Highly purified C3bINA cleaved neither free C3b nor free C4b if trace amounts of contaminating beta1H were removed from these proteins with anti-beta1H Sepharose. However, in the presence of highly purified beta1H and C3bINA, both C3bIna, both C3b and C4b were cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their cleaved. Incubation of native C3 or C4 with C3bINA and beta1H had no effect on their structure. The action of C3bINA and beta1H on C3b produced two fragments of the alpha1-chain which did not dissociate without reduction of the molecule. These fragments have mol wt of 67,000 and 40,000 daltons. The action of C3bINA and beta1H on C4b resulted in cleavage of the alpha'-chain giving rise to the 150,000-dalton C4c and the 49,000-dalton C4d fragments which dissociated without reduction. To produce from C3b the immunochemically defined C3c and C3d, fragments, the action of an additional serum enzyme appears to be required, the effect of which can be mimicked by trypsin.

Opsonization of bacteroides by the alternative complement pathway reconstructed from isolated plasma proteins.

Opsonization of clinical isolates of B. fragilis and B. thetaiotaomicron with the six isolated proteins of the alternative complement pathway under physiological conditions resulted in considerable C3 deposition on the bacterial surfaces. The time course of C3 deposition was similar to that observed in EGTA-serum; however, the magnitude of C3 deposition was twofold greater in EGTA-serum. Opsonization of the bacteria with the isolated alternative pathway proteins failed to promote adherence, uptake, or killing by polymorphonuclear leukocytes, whereas opsonization of the bacteria with EGTA-serum facilitated these events. The difference in opsonic capacity of isolated proteins and EGTA-serum was not related to the quantitative difference in C3 deposition, because repeated opsonization of the bacteria with isolated proteins resulting in C3 deposition comparable to that observed in EGTA-serum only minimally increased adherence of the bacteria to polymorphonuclear leukocytes. SDS-PAGE and autoradiographic analysis of C3 extracted from bacteria opsonized with isolated proteins or EGTA-serum using methylamine and SDS demonstrated that the predominant form of C3 bound by ester bonds under both sets of conditions was iC3b. A low molecular weight C3 cleavage fragment was detected in extracts from bacteria opsonized with isolated proteins, but it accounted for only a minor fraction of the bound C3. The results of our study demonstrate that the early phase of opsonization involving activation of the alternative pathway by B. fragilis and B. thetaiotaomicron and resultant C3 deposition on the bacterial surfaces does not require auxiliary serum factors, but the effector phase of opsonization of these bacteria involving recognition of bacteria-bound C3 by polymorphonuclear leukocytes and the induction of phagocytosis and intracellular killing is dependent on such factors. Natural IgM antibodies serve as auxiliary factors is opsonization of B. thetaiotaomicron by the alternative pathway, whereas additional serum factors are required for alternative pathway-mediated opsonization of B. fragilis.

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

Properdin- and nephritic factor-dependent C3 convertases: requirement of native C3 for enzyme formation and the function of bound C3b as properdin receptor.

Two complex enzymes were assembled that both converted C3 to C3b, one consisting of activated properdin (P), native C3, proactivator (PA) and proactivator convertase (PAase), and the other of nephritic factor (NF) and the same three cofactors. By maintaining a critical concentration of PAase, the P-C3 convertase and the NF-C3 convertase were shown to function efficiently without formation of the C3b-feedback enzyme. The former two enzymes are distinct from the C3b-dependent C3 convertase in that they utilize native C3 instead of C3b and PA in an apparently uncleaved form. The P- and NF-C3 convertase express maximal activity within approximately 10 min at 37 degrees C and decay with a half-life of 35 min at 37 degrees C, which is in contradistinction to the reported lability of the C3b-feedback enzyme. P- and NF-C3 convertases are inhibited by their product C3b, which may constitute a heretofore unknown control of the alternative pathway. A direct physical interaction of P with native C3 and C3b was demonstrated by agglutination of C3b-bearing erythrocytes and by agglutination inhibition. Bound C3b thus constitutes the only known receptor of P and may fulfill an important localizing function for P and the P-C3 convertase in vivo. Although P and NF form functionally similar enzymes, they act independently of each other and are apparently immunochemically unrelated proteins.

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75.

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.

Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process.

Tumor necrosis factor (TNF) alpha is a cytokine that has potent immune regulatory functions. To assess the potential role of this cytokine in the early development of autoimmunity, we investigated the effect of TNF on the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice, a spontaneous murine model for autoimmune, insulin-dependent type I diabetes. Treatment of newborn female NOD mice with TNF every other day for 3 wk, led to an earlier onset of disease (10 versus 15 wk of age in control mice) and 100% incidence before 20 wk of age (compared to 45% at 20 wk of age in control phosphate-buffered saline treated female mice). In contrast, administration of an anti-TNF monoclonal antibody, TN3.19.12, resulted in complete prevention of IDDM. In vitro proliferation assays demonstrated that mice treated with TNF developed an increased T cell response to a panel of beta cell autoantigens, whereas anti-TNF treatment resulted in unresponsiveness to the autoantigens. In addition, autoantibody responses to the panel of beta cell antigens paralleled the T cell responses. The effects mediated by TNF appear to be highly age dependent. Treatment of animals either from birth or from 2 wk of age had a similar effect. However, if treatment was initiated at 4 wk of age, TNF delayed disease onset. These data suggest that TNF has a critical role in the early development of autoimmunity towards beta-islet cells.

Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.

The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.