(CANCER RESEARCH 46, 5077-5083, October 1986] Macrophage Tumoricidal Activity Induced By Human C-Reactive Protein1 Kamyar Zahedi and Richard F. Mortensen Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 ABSTRACT Purified C-reactive protein (CRP), the prototypical acute phase reac- tant of humans, activated inflammatory mouse macrophages to a tumor- icidal state. The activation by CRP was not due to small amounts of contaminating lipopolysaccharide. CRP at 10 fig/ml induced significant tumoricidal capacity in resident macrophages; the mouse macrophage cell lines PUS 1.8, RAW 264.7, and J774; as well as elicited macrophages from two lipopolysaccharide nonresponder strains, C3H/HeJ and C57BL/10SC. Macrophages obtained from bone marrow-derived mono- cytes grown in vitro and exúdate macrophages depleted of T-cells were also readily activated by Mg/ml amounts of CRP. Removal of CRP from culture medium using anti-CRP antibodies or phosphorylcholine-agarose beads abrogated the induction of tumoricidal activity. CRP acted inde pendently of both lymphokines and lipopolysaccharide. Therefore, CRP may serve as a physiologically relevant macrophage activator, contrib uting to the heightened nonspecific host resistance associated with the early stages of a systemic inflammatory response. INTRODUCTION Mononuclear phagocytic cells are one of the major effector populations of innate, nonspecific host resistance. Activated macrophages have been shown to play a critical role in resist ance to intracellular microbial infections and are thought to limit growth of neoplastic cells (1,2). During a systemic inflam matory response, substantial monocytosis occurs that is often accompanied by the migration of the monocytes into inflam matory lesions (3,4). One of the hallmarks of the early or acute stage of a systemic inflammatory response is the rapid increase in the rate of synthesis by hepatocytes of a group of blood proteins termed APR2 (5). The classical APR of humans is CRP which increases in concentration from 0.1 to 300-500 /¿g/ ml within 24 h in response to tissue injury and especially to bacterial and viral infections (reviewed in Refs. 6 and 7). CRP is selectively deposited at sites of tissue damage which may also either contain or develop a monocyte-macrophage infÃ-ltrate(8, 9). Whether CRP directly affects macrophage functions has not been examined. The molecular properties, binding specificities, and certain i/i vitro activities mediated by CRP are consistent with a role for it as a mediator of nonspecific immunity. CRP is a pen- traxin, consisting of five identical noncovalently linked M, 21,500 subunits (10). The complete amino acid sequence of CRP has been determined (11, 12) along with its nucleotide sequence from the complementary DNA (13). The structural gene for CRP has been mapped to chromosome 1 (13). CRP has a primary Ca2+-dependent binding site for PC (14) and a Received 2/18/86; accepted 6/5/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This project was funded in part by USPHS Grants CA 30015 and AM 33296. Presented in a preliminary form at the 21st National Meeting of the RES Society, Montreal, Canada, October 14-17, 1984. Submitted by Kamyar Zahedi in partial fulfill mum of the requirements for a Ph.D. from The Ohio State University. 2The abbreviations used are: APR, acute phase reactant(s); CRP, C-reactive protein; MDP, muramyl dipeptide; LK, lymphokine; PC, phosphorylcholine; TBS, Tris-buffered saline; LPS, lipopolysaccharide; FBS, fetal bovine serum; PEC, peritoneal exúdate cells; MAP, macrophage activating factor, DMEM, Dulbecco's modified Eagle's medium; | 'H]d I lui, tritiated thymidine; HEPES, 4- (2-hydroxyethyl)-l-piperazineethanesulfonicacid; ELISA, enzyme linked immu- nosorbent assay; SDS, sodium dodecyl sulfate. second binding site for certain polycations (15). The binding and structural properties of CRP have been conserved through out vertebrate evolution, although it is not an APR in some species (7). CRP is opsonic and facilitates phagocytosis by both macrophages (16, 17) and neutrophils (18); it is also a potent activator of the classical complement cascade (19, 20) but restricts alternative pathway activation (21). CRP also binds to a subset of the Fc receptor bearing lymphocyte populations and affects several in vitro T-cell and B-cell lymphocyte activities (22, 23). Although these activities are associated with host defense, a clearly defined role for CRP in preimmune host resistance remains unknown. Cells of the monocyte-macrophage lineage selectively destroy tumor cells when activated by either LKs, such as 7-interferon (24, 25), or bacteria or their products such as MDP (26) or LPS (27). Macrophages are much more efficiently activated by either LKs or MDP delivered via liposomes than by the same substances in their free form (28). Recently, Deodhar et al. (29, 30) demonstrated that purified human CRP encapsulated in liposomes was effective in reducing the growth of métastasesof mouse tumors originating from different tissues. The liposomes containing CRP also increased macrophage tumoricidal capac ity in vitro (30). In this study we find that purified human CRP in its free form was capable of mediating activation of inflam matory macrophages to a tumoricidal state without any appar ent requirement for a LK. We also show that CRP activates macrophages under conditions which largely exclude partici pation by small amounts of LPS. The findings suggest a phys iologically relevant mechanism for macrophage activation dur ing the early stage of inflammation. MATERIALS AND METHODS Mice. Specific pathogen free female C3H/HeN (LPS responder), C3H/HeJ (LPS low responder), and A/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Female C57BL/6, C57BL/ 10SN, and C57BL/10ScN (LPS low responder) mice were purchased from HarÃ-anSprague-Dawley (Indianapolis, IN). The mice were housed in a Bioclean model PCS-80 filter chamber (Hazelton Laboratories, Bethesda, MD) with an air flow of 25 ft2/min. Mice were used at 6-12 weeks of age. The housing was essential to reduce the background macrophage tumoricidal activity (31). Tissue Culture Media and Reagents. DMEM and RPMI 1640 (M. A. Bioproducts, Walkersville, MD) were supplemented with 10% FBS (Hyclone, Inc., Logan, UT), 2 IHML-glutamine, and gentamicin sulfate (25 »ig/ml)and buffered with 10 IHM HEPES (U.S. Biochemicals, Cleveland, OH). Polymyxin B sulfate (Sigma Chemical Co., St. Louis, MO) was dissolved in pyrogen free water (Travenol Laboratories, Inc., Deerfield, IL). Detoxi-Gel, obtained from Pierce Biochemicals (Rock- ford, IL), was used for binding and removal of LPS. Phenol extracted LPS from Escherichia coli (strain 055:B5) and concanavalin A were obtained from Sigma. Affinity purified goat anti-human CRP was obtained from Jackson Immunoresearch (Avondale, PA) and the IgG fraction was conjugated onto Sepharose 4B (Pharmacia). Cells. Target cells used were: CAKI-1, a human renal carcinoma cell line; L-929, transformed murine fibre»blasts; and the P815 mastocy- toma, all from the American Type Culture Collection (Rockville, MD). Primary expiants of human foreskin fibroblasts were obtained from the cell culture laboratory, Department of Pathology, Ohio State Univer sity. Mouse monocyte-macrophage cell lines P388Di, PUS 1.8, J774 5077 Research. on November 5, 2021. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from