Macrophage Migration Inhibitory Factor Is a Critical Mediator of The
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11383–11388, September 1998 Medical Sciences Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria (T lymphocyteycytokineytoxic shock syndrome toxin 1ysuperantigensyseptic shock) THIERRY CALANDRA*, LORI A. SPIEGEL,CHRISTINE N. METZ, AND RICHARD BUCALA† Laboratory of Medical Biochemistry, The Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030 Communicated by Maclyn McCarty, The Rockefeller University, New York, NY, July 20, 1998 (received for review March 25, 1998) ABSTRACT Discovered in the early 1960s as a T cell acerbates LPS lethality, whereas neutralizing anti-MIF anti- cytokine, the protein mediator known as macrophage migra- bodies fully protect mice from endotoxic shock. Similar ob- tion inhibitory factor (MIF) has been found recently to be a servations have been made recently in a mouse model of pituitary peptide released during the physiological stress Escherichia coli peritonitis (T.C., unpublished observations). response, a proinflammatory macrophage cytokine secreted Studies of MIF expression by mouse and human T lymphocytes after LPS stimulation, and a T cell product expressed as part also established that MIF is a proinflammatory T cell cytokine of the antigen-dependent activation response. We report that is required for T cell activation and antibody production herein that MIF also plays a critical role in the innate host by B cells (10). Finally, the critical regulatory role played by response to staphylococcal and streptococcal exotoxins. In MIF was underscored by the finding that glucocorticoids at low RAW 264.7 or elicited mouse peritoneal macrophages, peak dose stimulated the production of MIF by macrophages and T MIF secretion was induced by concentrations of the staphy- cells, the first such response ascribed to glucocorticoids to date lococcal toxic shock syndrome (TSS) toxin 1 (TSST-1) and the (6, 10). Importantly, MIF has been shown to function to streptococcal pyrogenic exotoxin A as low as 10 pgyml. control or ‘‘counter-balance’’ the anti-inflammatory and im- Moreover, dose-response studies of splenocyte cytokine pro- munosuppressive effects of glucocorticoids on macrophages duction showed that lower concentrations of TSST-1 (10 and T cells (6, 10, 11). pgyml) were needed to release MIF than to induce interleukin The proportion of severe infections and septic shock caused 2 or interferon-g secretion (1 ngyml). We also studied the by Gram-positive bacteria has increased markedly in recent effect of neutralizing anti-MIF antibodies on TSST-1-induced years, such that these pathogens now account for 40–50 lymphocyte proliferation and lethal toxic shock. Pretreatment percent of all cases of septic shock occurring in the intensive of C57BLy6 mice with anti-MIF antibody 2 hr before TSST-1 care setting (12). Staphylococcal and streptococcal toxic-shock injection prevented spleen enlargement and reduced by 50% syndromes (TSS) and viridans streptococcal infections accom- the proliferation of splenocytes measured ex vivo. In a lethal panied by shock or the adult respiratory distress syndrome are mouse model of TSST-1-induced shock, anti-MIF antibody examples of the fulminant and often fatal complications of increased survival from 8% to 54% (P < 0.0001). These studies Gram-positive sepsis. In contrast to Gram-negative septic indicate that Gram-positive exotoxins are extremely potent shock, very little is known about the pathophysiology of inducers of MIF secretion and establish a critical role for MIF Gram-positive infections leading to septic shock. In the case of and the macrophage in the pathogenesis of the TSSs and in the TSS for instance, staphylococcal and streptococcal exotoxins innate immune response. appear to cause a massive activation of macrophages and T lymphocytes, which leads to the production of high levels of proinflammatory cytokines (13–18). Many Gram-positive bac- The biochemical nature and biological function of the protein teria do not produce exotoxins however, and they cause shock known as macrophage migration inhibitory factor (MIF), by mechanisms that remain to be fully unraveled. which was described originally to beaTlymphocyte product Given the central regulatory role of MIF in both the that inhibited macrophage migration, remained enigmatic for macrophage and the T cell limbs of the acute inflammatory almost three decades (1–4). Recent findings, however, have and immune responses, we have investigated the extent as well ascribed a pivotal role to MIF within the immune and endo- as the role of MIF expression in the host response to Gram- crine systems. First, the sequence structure of a newly identi- positive exotoxins. In this study, we report that the TSS toxin-1 fied protein that was released by pituitary cells in a hormone- (TSST-1) and the streptococcal pyrogenic exotoxin A (SPEA) like fashion was found to be that of MIF (5–7). Second, are very potent inducers of MIF production by immune cells monocytes and macrophages that had previously been consid- and that MIF is an important mediator of lymphocyte activa- ered to be the target of MIF action, were observed to be a tion and toxic shock triggered by these toxins. significant source of MIF after stimulation with endotoxin (lipopolysaccharide, or LPS) or with the cytokines tumor necrosis factor a (TNFa) and interferon-g (IFNg) (8). MIF MATERIALS AND METHODS also was described to mediate certain pro-inflammatory ef- Reagents. TSST-1 and streptococcal pyrogenic exotoxin A fects, stimulating macrophages to produce TNFa and nitric (SPEA) were obtained from Toxin Technology (Sarasota, FL). oxide when given in combination with IFNg (8, 9). Like TNFa b and IL-1 , MIF plays a central role in the host response to Abbreviations: MIF, macrophage migration inhibitory factor; LPS, endotoxemia. Coinjection of recombinant MIF and LPS ex- lipopolysaccharide; SPEA, streptococcal pyrogenic exotoxin A; TSS, toxic-shock syndrome; TSST-1, TSS toxin 1; IL, interleukin; IFN, The publication costs of this article were defrayed in part by page charge interferon; TNF, tumor necrosis factor. *Present address: Division of Infectious Diseases, Department of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Internal Medicine, Centre Hospitalier Universitaire Vaudois, CH- accordance with 18 U.S.C. §1734 solely to indicate this fact. 1011 Lausanne, Switzerland. © 1998 by The National Academy of Sciences 0027-8424y98y9511383-6$2.00y0 †To whom reprint requests should be addressed. e-mail: rbucala@ PNAS is available online at www.pnas.org. picower.edu. 11383 Downloaded by guest on September 24, 2021 11384 Medical Sciences: Calandra et al. Proc. Natl. Acad. Sci. USA 95 (1998) According to the manufacturer, the toxins were $95% pure described (8). MIF concentrations in cell-conditioned media and the LPS content of all the batches used ranged between were quantified by a sandwich ELISA using a monoclonal 0.02–0.075 endotoxin unit (equal to 2–7.5 pg of LPS) per mg anti-mouse MIF capture antibody, a polyclonal rabbit anti- of proteins. TSST-1 did not react with antibodies to the mouse MIF detector, and purified mouse recombinant MIF as staphylococcal enterotoxins A, B, C, D, and E or to the standard (6, 11). Samples were analyzed in triplicate and the exfoliative toxin A. SPEA did not react with antibodies to the results expressed as mean 6 SEM of two or three separate streptococcal pyrogenic exotoxins B and C. The toxins were experiments. resuspended in pyrogen-free water at a concentration of 1 Effect of Anti-MIF Antibody on Splenocyte Proliferation in mgyml, aliquoted and stored at 280°C. Anti-IL-2 mAb was Vitro. BALByc splenocytes (4 3 105 cells) were cultured in from Genzyme. D-Galactosamine, carbenicillin, Tween-20 96-well flat bottom tissue culture plates (Linbro) in RPMI were obtained from Sigma. Gentamicin was from GIBCO. containing 1% homologous mouse serum. Cells were incu- Thioglycollate broth (Difco) was prepared according to the bated for 72 hr with TSST-1 at 0.01, 1, 10, and 100 ngyml in the manufacturer’s recommendation, autoclaved, and stored pro- presence of nonimmune IgG, anti-MIF IgG (each at 50 mgyml) tected from light at room temperature. Horseradish peroxi- or anti-IL-2 IgG (5 mgyml). Cells then were pulsed for 6 hr with dase-conjugated goat anti-rabbit antibody was purchased from [3H]thymidine (1 mCi per well; 1 Ci 5 37 GBq), harvested and 9 9 Pierce and 4-chloro-1-naphthol and stabilized 3,3 ,5,5 - the incorporation of [3H]thymidine into DNA measured by tetramethylbenzidene substrate for horseradish peroxidase liquid scintillation counting in a microplate reader (TopCount, were from Promega. Polyclonal anti-MIF serum was generated Packard). by immunizing New Zealand White rabbits (Hare Marland, Effect of Anti-MIF Antibody on Splenocyte Proliferation in Hewitt, NJ) with purified, mouse recombinant MIF as de- Vivo. Eight to 10 week-old (19–21 g) female C57BLy6 mice scribed (8). Anti-MIF and nonimmune IgG were isolated from (Charles River Breeding Laboratories) were housed in groups serum by protein-G affinity chromatography following the of 5 or 10 mice per cage with free access to food and water and manufacturer’s recommendations (Pharmacia). were acclimatized for at least 5 days before experimentation. Cells. Mouse RAW 264.7 macrophages and AtT-20 anterior Groups of three mice were injected i.p. with 200 ml of anti-MIF pituitary cells were from the American Type Culture Collec- or nonimmune rabbit serum 2 hr before i.p. injection of tion (Manassas, VA). Thioglycollate-elicited peritoneal mac- m y y TSST-1 (average dose per mouse: 20 g, i.e., 1 mg kg body rophages were obtained from BALB c mice that were injected weight) diluted into 0.2 ml of saline. After 72 hr, mice were i.p. with 2 ml of sterile thioglycollate broth. Seventy-two hours euthanized by CO2 asphyxiation and the spleens removed and after injection, cells were harvested by lavage of the peritoneal weighed.