(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 second binding site for certain polycations (15). The binding and structural properties of CRP have been conserved through Purified C-reactive protein (CRP), the prototypical acute phase reac- out vertebrate evolution, although it is not an APR in some tant of humans, activated inflammatory mouse macrophages to a tumor- species (7). CRP is opsonic and facilitates phagocytosis by both icidal state. The activation by CRP was not due to small amounts of macrophages (16, 17) and neutrophils (18); it is also a potent contaminating lipopolysaccharide. CRP at 10 fig/ml induced significant tumoricidal capacity in resident macrophages; the mouse macrophage activator of the classical complement cascade (19, 20) but cell lines PUS 1.8, RAW 264.7, and J774; as well as elicited macrophages restricts alternative pathway activation (21). CRP also binds to from two lipopolysaccharide nonresponder strains, C3H/HeJ and a subset of the Fc receptor bearing lymphocyte populations and C57BL/10SC. Macrophages obtained from bone marrow-derived mono- affects several in vitro T-cell and B-cell lymphocyte activities cytes grown in vitro and exúdate macrophages depleted of T-cells were (22, 23). Although these activities are associated with host also readily activated by Mg/ml amounts of CRP. Removal of CRP from defense, a clearly defined role for CRP in preimmune host culture medium using anti-CRP antibodies or phosphorylcholine-agarose resistance remains unknown. beads abrogated the induction of tumoricidal activity. CRP acted inde Cells of the monocyte-macrophage lineage selectively destroy pendently of both lymphokines and lipopolysaccharide. Therefore, CRP tumor cells when activated by either LKs, such as 7-interferon may serve as a physiologically relevant macrophage activator, contrib uting to the heightened nonspecific host resistance associated with the (24, 25), or bacteria or their products such as MDP (26) or early stages of a systemic inflammatory response. 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, INTRODUCTION 30) demonstrated that purified human CRP encapsulated in liposomes was effective in reducing the growth of métastasesof Mononuclear phagocytic cells are one of the major effector mouse tumors originating from different tissues. The liposomes populations of innate, nonspecific host resistance. Activated containing CRP also increased macrophage tumoricidal capac macrophages have been shown to play a critical role in resist ity in vitro (30). In this study we find that purified human CRP ance to intracellular microbial infections and are thought to in its free form was capable of mediating activation of inflam limit growth of neoplastic cells (1,2). During a systemic inflam matory macrophages to a tumoricidal state without any appar matory response, substantial monocytosis occurs that is often ent requirement for a LK. We also show that CRP activates accompanied by the migration of the monocytes into inflam macrophages under conditions which largely exclude partici matory lesions (3,4). One of the hallmarks of the early or acute pation by small amounts of LPS. The findings suggest a phys stage of a systemic inflammatory response is the rapid increase iologically relevant mechanism for macrophage activation dur in the rate of synthesis by hepatocytes of a group of blood proteins termed APR2 (5). The classical APR of humans is ing the early stage of inflammation. CRP which increases in concentration from 0.1 to 300-500 /¿g/ ml within 24 h in response to tissue injury and especially to MATERIALS AND METHODS bacterial and viral infections (reviewed in Refs. 6 and 7). CRP is selectively deposited at sites of tissue damage which may also Mice. Specific pathogen free female C3H/HeN (LPS responder), either contain or develop a monocyte-macrophage infíltrate(8, C3H/HeJ (LPS low responder), and A/J mice were purchased from 9). Whether CRP directly affects macrophage functions has not The Jackson Laboratory (Bar Harbor, ME). Female C57BL/6, C57BL/ been examined. 10SN, and C57BL/10ScN (LPS low responder) mice were purchased The molecular properties, binding specificities, and certain from HaríanSprague-Dawley (Indianapolis, IN). The mice were housed in a Bioclean model PCS-80 filter chamber (Hazelton Laboratories, i/i vitro activities mediated by CRP are consistent with a role Bethesda, MD) with an air flow of 25 ft2/min. Mice were used at 6-12 for it as a mediator of nonspecific immunity. CRP is a pen- weeks of age. The housing was essential to reduce the background traxin, consisting of five identical noncovalently linked M, macrophage tumoricidal activity (31). 21,500 subunits (10). The complete amino acid sequence of Tissue Culture Media and Reagents. DMEM and RPMI 1640 (M. CRP has been determined (11, 12) along with its nucleotide A. Bioproducts, Walkersville, MD) were supplemented with 10% FBS sequence from the complementary DNA (13). The structural (Hyclone, Inc., Logan, UT), 2 IHML-glutamine, and gentamicin sulfate gene for CRP has been mapped to chromosome 1 (13). CRP (25 »ig/ml)and buffered with 10 IHM HEPES (U.S. Biochemicals, has a primary Ca2+-dependent binding site for PC (14) and a Cleveland, OH). Polymyxin B sulfate (Sigma Chemical Co., St. Louis, MO) was dissolved in pyrogen free water (Travenol Laboratories, Inc., Received 2/18/86; accepted 6/5/86. Deerfield, IL). Detoxi-Gel, obtained from Pierce Biochemicals (Rock- 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 ford, IL), was used for binding and removal of LPS. Phenol extracted accordance with 18 U.S.C. Section 1734 solely to indicate this fact. LPS from Escherichia coli (strain 055:B5) and concanavalin A were 1This project was funded in part by USPHS Grants CA 30015 and AM 33296. obtained from Sigma. Affinity purified goat anti-human CRP was Presented in a preliminary form at the 21st National Meeting of the RES Society, obtained from Jackson Immunoresearch (Avondale, PA) and the IgG 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. fraction was conjugated onto Sepharose 4B (Pharmacia). 2The abbreviations used are: APR, acute phase reactant(s); CRP, C-reactive Cells. Target cells used were: CAKI-1, a human renal carcinoma cell protein; MDP, muramyl dipeptide; LK, lymphokine; PC, phosphorylcholine; line; L-929, transformed murine fibre»blasts;and the P815 mastocy- TBS, Tris-buffered saline; LPS, lipopolysaccharide; FBS, fetal bovine serum; toma, all from the American Type Culture Collection (Rockville, MD). PEC, peritoneal exúdate cells; MAP, macrophage activating factor, DMEM, Dulbecco's modified Eagle's medium; | 'H]d I lui, tritiated thymidine; HEPES, 4- Primary expiants of human foreskin fibroblasts were obtained from the (2-hydroxyethyl)-l-piperazineethanesulfonicacid; ELISA, enzyme linked immu- cell culture laboratory, Department of Pathology, Ohio State Univer nosorbent assay; SDS, sodium dodecyl sulfate. sity. Mouse monocyte-macrophage cell lines P388Di, PUS 1.8, J774 5077 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. CRP-MEDIATED MACROPHAGE ACTIVATION A.I, and RAW 264.7 were also obtained from American Type Culture substrate. The CRP concentration was determined by either radial Collection. The hybridoma HO-13-4 which produces anti-Thy-1.2 immunodiffusion using a goat anti-human CRP or by a competitive (IgM) American Type Culture Collection has been characterized pre ELISA using affinity purified goat anti-human CRP (Jackson Inumi viously (32). All cell lines were Mycoplasma free and were monitored noresearch). The purity of CRP was assessed by SDS-polyacrylamide throughout these experiments. gel electrophoresis under reducing conditions using a 4.0% stacking gel Spleen Cell Culture Supernatants. Lymphocyte culture supernatants and a 12.0% resolving gel. The protein in these preparations was 97% containing MAP were generated by incubating 10* C3H/HeN spleen CRP based on reactivity with antibody in the competitive ELISA. cells/ml with concanavalin A (2.5 jig/ml) for 72 h in RPMI 1640 Assay for LPS. The LPS (endotoxin) content of CRP and tissue containing 25 ¿IM2-mercaptoethanol and 5% FBS. A single MM culture reagents was measured by the chromogenic Limulus amebocyte preparation was used for these studies. lysate assay (LAL; M. A. Bioproducts) using the LPS standard supplied Peritoneal Macropahges. PEC were elicited by i.p. injection of 2.5 with the kit. The four CRP preparations used in these studies had 0.05- ml of sterile 10% proteose peptone (Difco Laboratories, Detroit, MI). to 0.10-ng/ml amounts of LPS; the eliciting agents and tissue culture PEC or resident macrophages were harvested by lavage using cold components had <0.05-ng/ml amounts of LPS. DMEM. Cells were washed three times in DMEM containing 10% Removal of CRP. CRP was removed from the culture medium by FBS. Monolayers of adherent cells were prepared by seeding 2.5 x 10s treatment with agarose beads (A-0.5m; Bio •Rad)conjugated with either cells/well in 96-well flat bottomed tissue culture plates (Microtest II; PC or affinity purified goat IgG anti-human CRP. Briefly, conjugated Falcon) and incubating for 4 h (37"C, 5% COi). Nonadherent cells agarose beads were added to culture medium containing CRP (40 Mg/ were removed by extensive (5-7 times), uniform washing with cold ml) at a volume of 50, 10, and 1% and incubated at 40'C for 24 h.