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We have previously extracted a protein from inflammatory mouse granuloma that fully protects normal mice against lethal Listeria monocytogenes infection. We now report that polyclonal antibodies directed against this protein react with a human urinary fraction that also provides full protection of normal mice against L. monocytogenes. Murine monoclonal antibodies completely inhibit the protective activity of the human urinary fraction. We have purified to apparent homogeneity a human glycoprotein of 43 kDa (HGP.43), pI = injection of C57BL/6 mice with Bio-Gel P-100 (Bio-Rad), the gel was withdrawn and the inflammatory cells were recovered by filtration through a 400-mesh screen. The cells were centrifuged (250 x g for 10 min), washed twice with ice-cold Dulbecco's modified Eagle's medium (DMEM), incubated (1 x 106 cells per ml, 0.5 ml per culture tube) for 24 hr, and washed to remove polymorphonuclear cells. Lewis tumor cells (3) were isolated from murine lung metastases and cultured in plastic Petri dishes in DMEM containing 10% (vol/vol) fetal calf serum at 3TC in an atmosphere of 7.5% C02/92.5% air.Antibodies. Polyclonal rabbit anti-mouse granuloma protein antibodies were prepared as described by Fontan et al. (1). Polyclonal antibodies against the human urinary protein were raised in rabbits by s.c. injection of 10 ,ug in Freund's complete adjuvant and twice-monthly booster injections. Immunoglobulins were purified by precipitation in (NH4)2SO4 at 33% saturation and DEAE-gel chromatography. Antibodies against human al-AGP were purified by means ofimmunoaffinity chromatography using pure a1-AGP (Sigma) coupled to CNBr-activated Sepharose. Monoclonal antibodies against the human urinary protein were prepared as follows. Female BALB/c mice were immunized by s.c. injection of 50 jug of a semipurified active human urinary fraction. Spleen cells were fused with the SP2/0 BALB/c myeloma cell line. Wells with hybrid growth were screened for reactivity with the urinary fraction by using an ELISA. Positive wells were subcloned twice by limiting dilution. One of the subclones (K52.5G2) was chosen for the neutralization test. This clone produces an IgG1 that was purified by (NH4)2SO4 precipitation and DEAE-gel chromatography. The supernatants and antibody-free controls were tested in mice challenged with a lethal inoculum ofL. monocytogenes.All the materials, solutions, and tissue culture media were free of endotoxin.Statistics. Results are expressed as the mean ± SEM. The significance of the differences between experimental groups was analyzed by Student's t test. A P value of <0.05 was considered significant. RESULTS Purification and Characterization of the Urinary Protein. After Amicon ultrafiltration concentration of the urines, the material was chromatographed on a DEAE-Sephacel anionexchange column and eluted with 0.2 and 1 M NaCl. All the immunostimulating activity was eluted with 0.2 M NaCI (Fig. 1). The active fractions were pooled, dialyzed, and loaded onto a Cibacron blue column to remove contaminat...

HGP92 has been shown to enhance in vitro and in vivo the bactericidal and tumoricidal activity of mouse macrophages. In this study we investigated the effect of HGP92 on the accumulation of cytokine mRNA in mouse inflammatory, peritoneal macrophages and the monocytic cell line J774. HGP92 significantly enhanced the level of cytokine mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, TNF-alpha and GM-CSF during the first 24 h of the incubation. This effect triggered by HGP92 was comparable to that obtained with lipopolysaccharide (LPS), which is a strong cytokine inducer. This accumulation of cytokine mRNA in macrophages was correlated with secretion of IL-6 and TNF-alpha in cell supernatant. The release of IL-6 was HGP92 concentration dependent over a range of 0.3-10 micrograms/ml with a maximum production obtained after a 24 h incubation of inflammatory macrophages with HGP92. This effect was shown not to be due to contamination of HGP92 by LPS since inflammatory macrophages from C57BL/6 mice were responsive to HGP92 pretreated with polymyxin B sulfate and unresponsive to heated HGP92. Stimulating activity of HGP92 was confirmed using macrophages from C3H/HeJ mice. These results suggest that HGP92 might modulate the immune responses by increasing cytokine production by macrophages.