Proc. Nail. Acad. Sci. USA Vol. 87, pp. 3348-3352, May 1990 Medical Sciences Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: The monocyte Western blot (Mycobacterium tuberculosis) R. S. WALLIS, M. AMIR-TAHMASSEB, AND J. J. ELLNER Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, OH 44106 Communicated by Frederick C. Robbins, January 29, 1990 ABSTRACT Infection with Mycobacterium tuberculosis in- ated by LPS, however, as mycobacteria lack this polysac- volves mononuclear phagocytic cells as hosts to intracellular charide. Furthermore, purified protein derivative-induced parasites, accessory cells in the induction of the immune production of IL-1 is unaffected by polymyxin, a cationic response, effector cells for mycobacterial killing, and targets of polypeptide antibiotic that binds to the lipid A moiety of LPS cytotoxic lymphocytes. When stimulated by whole mycobacte- and inactivates most of its biologic activities (3, 4), thus also ria or various mycobacterial preparations, monocytes and excluding a contaminating role for LPS. The specific com- macrophages produce the cytokines interleukin 1 and tumor ponents of mycobacteria responsible for induction of mono- necrosis factor, which possess multiple functions, including cyte cytokine production are not known. In this series of immune induction, and may be responsible for the fever and experiments, we have adapted the technique of Western blot cachexia prominent in tuberculosis. To identify mycobacterial analysis to study monocyte activation and have identified proteins that may directly activate production of these cyto- protein fractions of M. tuberculosis at molecular weights of kines, culture filtrate ofM. tuberculosis that had been subjected approximately 46,000 and 20,000 that result in monocyte to gel electrophoresis and transferred to nitrocellulose paper expression of IL-1 and TNF. Corresponding fractions acti- was used to stimulate monocyte production of cytokines. Frac- vated T lymphocytes from healthy donors. The unique ca- tions representing molecular weights of 46,000 and 20,000 pacity to stimulate both mononuclear phagocytes and T consistently induced both interleukin 1 and tumor necrosis lymphocytes may define particularly immunogenic microbial factor. The magnitude of the monocyte responses to these products. fractions was similar to that to intact mycobacteria or optimal concentrations of lipopolysaccharide. This stimulatory effect METHODS was not due to contamination with either bacterial lipopoly- saccharide or mycobacterial lipoarabinomannan, as it was Antigens. M. tuberculosis strain H37Rv was cultured in abolished by digestion with Streptomyces griseus protease but Proskauer-Beck medium. After 4-6 weeks, cells were re- was unaffected by ammonium sulfate precipitation, preincuba- moved by sedimentation followed by filtration with a 0.4-gm tion with polymyxin B, or depletion of lipoarabinomannan by membrane. The filtrate was dialyzed against water using a immunoaffinity chromatography. Proteins identified by this Spectra/Por 2 membrane (Spectrum Medical Industries), system may have considerable potential as immunogens, as the lyophilized, and resuspended in water. A protein-enriched fraction was prepared by precipitation in 50% saturated capacity to directly stimulate mononuclear phagocyte produc- (NH4)2SO4, followed by resuspension of the precipitate and tion of cytokines is an essential property of adjuvants. dialysis against water. In some experiments, the culture filtrate was further purified by immunoabsorbent chromatog- Mononuclear phagocytes serve multiple functions in the raphy using monoclonal antibody to M. tuberculosis lipoara- pathophysiology of tuberculosis: host to an intracellular binomannan (LAM) (kindly provided by Patrick Brennan, parasite, accessory cell for induction of the immune re- Colorado State University) coupled to Sepharose with cyan- sponse, effector cell for mycobacterial killing, and target for ogen bromide. After repeated passage through the antibody killing by other cytotoxic cells. Activation of this complex column, the filtrate was concentrated over a Centricon filter. network hinges on the expression of mycobacterial antigens The protein content of each preparation was determined by on the surface ofinfected cells and the appropriate release by a colorimetric assay (Bio-Rad). In some experiments, the infected cells of the cytokines necessary for immune induc- anti-LAM treated M. tuberculosis filtrate was then digested tion. with a 5-fold excess of nonspecific protease type XIV from Mycobacteria are potent inducers of monocyte production Streptomyces griseus (Sigma) for 18 hr. ofthe cytokines interleukin 1 (IL-1) and tumor necrosis factor Gel Electrophoresis. Antigen (500 gg of protein or, in the (TNF) (1, 2). This is not dependent on the presence of intact case of the protease-treated preparation, the products of microbial organisms, as we have previously demonstrated digestion of 500 ug of protein) was mixed with an equal cytokine induction by the dialyzed filtrate ofMycobacterium volume of reducing sample buffer [4% SDS/20% (vol/vol) tuberculosis culture medium and by the protein-enriched glycerol/10% 2-mercaptoethanol/1.5% Tris HCI, pH 6.75], fraction of that medium (purified protein derivative) (2). The heated to 1000C for 2 min, and applied to a 7 x 10 cm 9% capacity for induction of IL-1 by soluble factors of other acrylamide gel. After electrophoresis, proteins were trans- bacteria has been thought mainly to reside in polysaccha- ferred to nitrocellulose paper, washed in phosphate-buffered rides; indeed, Escherichia coli lipopolysaccharide (LPS), the saline (PBS), and stained with Aurodye (Janssen). The ni- prototypic agent used to induce IL-1 release in vitro, is active trocellulose paper was cut into 2-mm horizontal strips, trans- in this respect even in nanogram concentrations. The capac- ferred to individual glass tubes, allowed to dry, and dissolved ity of purified protein derivative to induce IL-1 is not medi- in 1 ml of dimethyl sulfoxide (Sigma) (5); 3 ml of 0.05 M The publication costs of this article were defrayed in part by page charge Abbreviations: IL-1, interleukin 1; TNF, tumor necrosis factor; LPS, payment. This article must therefore be hereby marked "advertisement" lipopolysaccharide; LAM, lipoarabinomannan; PHS, pooled human in accordance with 18 U.S.C. §1734 solely to indicate this fact. serum; FCS, fetal calf serum. 3348 Downloaded by guest on September 27, 2021 Medical Sciences: Wallis et aL Proc. Natl. Acad. Sci. USA 87 (1990) 3349 Na2CO3 (pH 9.6) was added dropwise with continuous agi- each dilution was placed in microtiter wells in replicates of tation. The precipitate was pelleted, washed once, resus- three. Female C3H/HeJ mice (8-10 weeks old) were sacri- pended in 0.5 ml ofRPMI 1640 medium, and frozen at -300C. ficed by cervical dislocation. Thymic tissue was passed Western Blotting. Nonspecific binding was inhibited by through stainless steel mesh and washed in complete RPMI incubation of nitrocellulose transfers in RPMI 1640 medium 1640 medium. Aggregates were removed by brief 1-g sedi- with 10o fetal calf serum (FCS) for 2 hr at 370C. Monoclonal mentation. Cells were suspended in complete RPMJ 1640 antibody to LAM was diluted 1:1000 in 1% bovine serum medium with 20o FCS, 50 p.M 2-mercaptoethanol, and albumin in PBS. After overnight incubation at room temper- phytohemagglutinin (2 pug/ml) (Burroughs Wellcome) at a ature, the nitrocellulose paper was washed in PBS/0.01% density of 15 x 106 cells per ml. One hundred microliters of Tween 80, and incubated overnight in alkaline phosphatase- (1 coupled anti-mouse immunoglobulin (Sigma) diluted 1:1000 the cell suspension was added to each well. [3H]Thymidine in bovine serum albumin (1% in PBS). Alkaline phosphatase p.Ci) was added during the final 8 hr of a 72-hr culture. Cells activity was detected with nitroblue tetrazolium and 5- were harvested with a semiautomated harvester, and [3H]- bromo4-chloro-3-indolyl phosphate. thymidine content was assessed by scintillation photometry. Cell Culture. Sixty microliters of each fraction of nitrocel- IL-1 activity in half-maximal units/ml was determined by lulose particles was placed in individual 2-ml tissue culture probit analysis. wells, and incubated for 1 hr in 0.5 ml of complete medium [RPMI 1640 medium (M.A. Bioproducts) with 2 mM L- RESULTS glutamine/gentamicin (100 Ag/ml)/15 mM Hepes] with 4% heat-inactivated pooled human serum (PHS) and polymyxin Culture filtrate ofM. tuberculosis that had been subjected to B (20 pg/ml). LPS (1 g/ml) and purified M. tuberculosis SDS/PAGE, transferred to nitrocellulose paper, cut into LAM (provided by Patrick Brennan), both with and without horizontal strips, dissolved in dimethyl sulfoxide, and pre- polymyxin B, and whole irradiated (4 x 106 rad; 1 rad = 0.01 cipitated in an aqueous buffer to produce a fine particle Gy) M. tuberculosis (50 Ag/ml) with polymyxin B served as suspension was used to stimulate monocytes in culture, in an controls. Blood mononuclear cells from tuberculin-negative adaptation ofthe T-cell Western blot technique developed by donors were obtained by density sedimentation over Ficoll/ Abou-Zeid et al. (5) and Young and Lamb (6). The quantity Hypaque (Pharmacia). Tissue culture grade 100-mm Petri of filtrate applied to the gel was selected to allow adequate dishes were coated with PHS and incubated at 37°C for 15 representation of proteins without overloading