Abram B. Stavitsky

Differential Contributions of C3, C5, and Decay-Accelerating Factor to Ethanol-Induced Fatty Liver in Mice

BACKGROUND AND AIMS The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.

In Vitro Studies of the Antibody Response

Publisher Summary This chapter describes the antibody response, beginning with the entrance of antigen into certain cells, to all the cellular events that culminate in the synthesis of circulating antibody. The chapter presents two methods for in vitro study of antibody response—maintenance of function of antibody-forming cells and assays of antibody and antigen. The chapter examines certain general factors that affect all aspects of the antibody response and influence interpretation of experimental data, including the chemical and physical nature and dosage of antigen; the age, strain, and species of experimental animal; the amounts and type of antibody produced; the methods utilized for the assay of antigen and antibody. The chapter describes the mechanism of antibody response. This mechanism involved uptake and fate of antigen and examined cells involved in the initiation of the antibody response. For this, an in vitro uptake of antigen followed by antibody synthesis in vivo is performed.

EVIDENCE FOR THE INSULIN-DIRECTED SPECIFICITY OF RABBIT ANTI-INSULIN SERUM

In the previous paper (2) a method was described for the immunological assay of microgram quantities of insulin. This method employed the reaction between rabbit antiserum to insulin and sheep erythrocytes conjugated with insulin. However, the applicability of the method to the measurement of insulin is obviously dependent upon the insulin-directed specificity of the antiserum which is employed in the assay. Therefore, the present experiments were designed to determine the specificity of rabbit antisera to crystalline beef insulin. In order to establish this specificity answers were sought to these questions: 1) To what extent will the insulin antisera react with contaminants that may be present in the crystalline beef insulin used for immunization? 2) Will these antisera react with proteins derived from beef plasma and other sources? 3) To what extent will the antisera to beef insulin react with insulin from other species? 4) Will the antisera interfere with the physiological action of insulin? Previous investigators have provided much information on some of these points. Lewis (3) and Barral and Roux (4) have shown that purified insulin is a different antigen from those in serum or crude extracts of pancreas. The immunological similarity of insulin prepared from several species has been demonstrated by Lewis (3) and also by Wasserman and Mirsky (5). Using the SchultzDale technique, Lewis (3) showed that guinea pigs can be sensitized with insulin from one species and shocked with insulin from another species. Wasserman and Mirsky (5) confirmed this in sensitization and complement fixation experiments. However, these experiments were qualitative in nature. Furthermore, the insulin preparations

In vitro and in vivo regulation by macrophage migration inhibitory factor (MIF) of expression of MHC-II, costimulatory, adhesion, receptor, and cytokine molecules

The secretion of macrophage migration inhibitory factor (MIF) is enhanced by inflammatory and other stimuli. MIF regulates innate and adaptive immune responses, but the mechanisms of this regulation are poorly understood. Our hypothesis was that MIF generated by these stimuli regulates these responses by modulating key molecular expression. This hypothesis was tested by adding greater than constitutive concentrations of recombinant MIF to cultures of various cell types and flow cytometric assay. MIF modulated surface expression of MHC-II, B7-2, CD40, CD40 ligand, ICAM-1 and Fcgamma, CR1/CR2, and IL-10 receptors and intracellular expression of IL-10, TNFalpha, and p40 (IL-12). MIF increased expression of B7-1 by B cells and CD40 L by T cells in spleens from Schistosoma mansoni-infected mice. Footpad injection of MIF reduced expression of MHC-II and CD40 by B cells in draining lymph nodes. Footpad injection of Mab to MIF reduced expression of B7-2 and CR1/CR2 by B cells and B7-2 by macrophages in these nodes. These data support our hypothesis.

Mechanics of antibody globulin synthesis by lymphoid tissue in vitro

Abstract Lymph nodes from immunized rabbits effect a net synthesis of antibody on a synthetic medium in vitro . Several amino acid analogs inhibit this synthesis and their effects are reversed by the natural amino acids. An optimal concentration of amino acids in the medium is required for antibody formation. On the basis of isotope experiments it was concluded that cellular proteins were not converted to antibody in vitro and antibody was derived largely, if not exclusively, from free amino acids. Similarities between antibody synthesis and protein synthesis in microorganisms and mammalian tissues were indicated.

Regulation of the in vitro anamnestic antibody response by cyclic AMP: II. Antigen-dependent enhancement by exogenous prostaglandins of the E series

Abstract Keyhole limpet hemocyanin was injected into the hind foot pads of rabbits. Six days later popliteal lymph node cell populations were prepared and induced in vitro with various amounts of KLH to produce an anamnestic antibody response. Previously it was observed that cholera enterotoxin which stimulates adenylate cyclase, or dibutyryl cyclic AMP, added for 0 to 24 hr to these cultures with 1 μg or more of KLH, enhanced antibody synthesis many-fold. In the current experiments these observations were confirmed. Moreover, the addition of optimal concentrations of prostaglandins E1 and E2—but not prostaglandin F1α—with 1 μg or more of KLH for 0 to 24 hr enhanced the ensuing antibody response two- to six-fold. When KLH was added for 0 to 24 hr and PGE1 or PGE2 for 72 to 120 hr, the antibody response was inhibited. Enhancement of antibody production required that the KLH-primed LNC first be exposed to KLH (1 to 100 μg) before addition of these PGs. The PG mediated elevation of antibody synthesis was Ca2+-dependent, whereas the induction of antibody synthesis by KLH was not. The LNC were fractionated on nylon fiber columns. The effluent cells—which consisted of approximately equal numbers of Ig+ (B) and Ig− (T) lymphocytes—were induced to produce antibody to the same extent as the unfractionated LNC. However, in contrast to the unfractionated cells, the antibody response of the effluent LNC was not enhanced by added PG. Elevation of the anamnestic antibody synthesis by PG showed the same requirements and specificity as regulation by CT and DbcAMP. Moreover, the magnitude of elevation of antibody synthesis by these different agents was approximately equal. Therefore, it was postulated that these three types of molecules modulate the antibody response via a common pathway involving cyclic AMP. A model is proposed in which KLH elevates cyclic AMP levels in a KLH-regulatory cell population causing production of soluble regulatory factor(s) which then enhance the antibody response by antigen induced T helper and /or B LNC. An antigen-induced elevation of PG may be required for the increase in cAMP, but the increase in cAMP is the crucial regulatory step.

Blockade of macrophage migration inhibitory factor (MIF) in Schistosoma japonicum‐infected mice results in an increased adult worm burden and reduced fecundity

Macrophage migration inhibitory factor (MIF), a cytokine produced by many cell types, modulates cellular and humoral immune responses. In schistosomiasis, ova in the portal circulation induce a delayed type hypersensitivity (DTH) that results in formation of hepatic granulomas (HG) which secrete MIF activity. Therefore, we hypothesized that endogenous MIF modulates immune responses in schistosomiasis. To test this hypothesis, Schistosoma japonicum‐infected mice were injected with rabbit IgG or neutralizing rabbit IgG antibody to MIF 4·5–6·5 week post infection when HG form and female worms are laying eggs. Compared with controls, 6·5–7‐week post‐infection, antibody‐treated mice had 1·7–3 times as many adult worms and half as many ova per worm pair in their livers. In contrast, antibody introduced before infection or 6–8 week post infection did not affect worm burden or fecundity. Thus, for the first time there is evidence that 4·5–6 week post‐infection endogenous MIF somehow mediates reduction of adult worm burden and promotes fecundity. Splenocytes and HG cells from antibody‐treated mice showed reduced intracellular expression of TNFα and/or IL‐10. We hypothesize that endogenous MIF enhances adult worm attrition by up‐regulating innate and adaptive immune responses by increasing expression of MHC‐II, co‐stimulatory, adhesion, receptor and cytokine molecules, and promotes fecundity by up‐regulating TNFα expression.

In vivo blockade of macrophage migration inhibitory factor prevents skin graft destruction after indirect allorecognition.

Background. The effector mechanisms that ultimately destroy transplanted tissues are poorly understood. In particular, it is not clear how CD4+ T cells primed to donor-derived determinants expressed on recipient MHC molecules (the indirect pathway) can mediate graft destruction in the absence of cognate recognition of peptide: MHC on the graft cells themselves. Macrophage migration inhibitory factor (MIF) inhibits macrophage movement and is a proinflammatory and regulatory cytokine known to be essential for development of delayed-type hypersensitivity reactions. Methods. To test whether MIF participates in graft destruction following indirect recognition, we studied rejection of MHC-II-deficient skin grafts placed on allogeneic SCID recipients adoptively transferred with naïve CD4+ T cells, and the recipients were treated with neutralizing anti-MIF monoclonal antibody or isotype control IgG. In this model graft rejection can only occur indirectly as the graft cells lack MHC II for recognition by the recipient CD4+ T cells. Results. We found that in vivo blockade of MIF inhibited indirect CD4+ cell-mediated skin graft destruction, and markedly reduced detectable macrophages within the grafts. The neutralizing anti-MIF antibody significantly inhibited alloreactive DTH but did not prevent T cell priming or interferon-&ggr; release by primed T cells. Conclusions. The results strongly implicate MIF as an active participant in skin graft destruction after indirect recognition and suggest that this effect is mediated through an inhibition of macrophage migration and/or function.

Preparation, characterization, and functions of rabbit lymph node cell populations. I. Preparation of KLH primed T and B memory cells with anti-fab' affinity columns.

Abstract Lymph node cells from rabbits primed with keyhole limpet hemocyanin (KLH) were separated into two populations by passage over goat anti-rabbit Fab′ (AFab′) columns. Cells with readily detected surface immunoglobulin (SIg) were retained on the column whereas those that passed through lacked SIg and receptors for C3. Adherent cells, eluted with purified rabbit IgG or normal rabbit serum (NRS) were mostly SIg+ and enriched for complement receptor lymphocytes (CRL) compared to the starting, or unfractionated population. Adherent LNC incorporated much less [ 3 H]thymidine than did non-adherent or unfractionated LNC when stimulated with Con A. When induced with AFab′, however, only adherent and unfractionated LNC incorporated [ 3 H]thymidine above background levels. Both non-adherent and adherent LNC from KLH primed rabbits showed a reduced capacity to synthesize anti-KLH antibody, total immunoglobulin, and total protein when induced with KLH in vitro . However, addition of KLH to a mixture of non-adherent and adherent LNC resulted in reconstitution of anti-KLH, total immunoglobulin, and total protein synthesis to the levels induced in unfractionated LNC. It was concluded that the non-adherent LNC consisted almost exclusively of T cells and the adherent LNC of B cells. By a variety of criteria the T cells were suitable for many types of studies of the antibody response. However, the B cells were not always suited for these studies.

Upregulation of DAF (CD55) on orbital fibroblasts by cytokines. Differential effects of TNF-ß and TNF-a

Purpose. Decay accelerating factor (DAF) and membrane cofactor protein (MCP) are membrane complement regulators that protect self cells from deposition of autologous C3b on their surfaces. CD59, a third downstream regulator of the cascade, prevents the assembly on self cells of autologous membrane-attack complexes. All three proteins are highly expressed on corneal and conjunctival epithelia, and are present in lower levels on multiple intraocular and adnexal cell types. The purpose of this study was to determine whether, and if so, how DAF, MCP and CD59 expression by ocular and adnexyl cells is modulated by cytokines. Methods. Primary cultures of orbital fibroblasts and corneal epithelial cells were incubated with TNF-a, TNF-ß, TGF-ß1, IFN-?, MIF or blocking anti-MIF mABs and extracts of the cells quantitated for DAF, MCP and CD59 by two-site immunoradiometric assays. Where inductions occurred, the kinetics of the increases, the effect of combining cytokines, and the effect of protein kinase-C inhibition were studied. Results. DAF expression on orbital fibroblasts was upregulated 6.3-, 3.7- and 4.2-fold by TGF-ß1, TNF-ß and IFN-?, respectively, but that its expression on corneal epithelial cells was minimally affected. These same (or other) cytokines did not significantly upregulate MCP or CD59. The cytokine-induced upregulation of DAF expression on orbital fibroblasts requires 24 hr for IFN-? or 48 hr for TGF-ß1 or TNF-ß, is dependent on new protein synthesis, and does not involve protein kinase-C activation. Conclusions. TGF-ß1-, TNF-ß- and IFN-?-mediated upregulation of DAF should serve to prevent complement-mediated injury to orbital fibroblasts in the course of ocular inflammation. The induction by TNF-ß rather than TNF-a contrasts with that on all other cell types studied.