Macrophage Tumoricidal Activity Induced by Human C-Reactive Protein1

(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 exÃºdate 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 mÃ©tastasesof 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 /Â¿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 infÃltrate(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 HarÃanSprague-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 Â»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 fibreÂ»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 exÃºdate 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 Â¿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 â€¢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. DMEM. Esterase staining and latex ingestion showed the presence of Samples were centrifuged at 13,000 rpm and the supernatants were 90 to 95% cytoplasmic esterase positive phagocytic cells. collected and filter sterilized. These supernatants were then assayed for Depletion of T-Cells. After adherence of PEC to the wells in the both CRP concentration and their ability to activate macrophages in microplate, the cells were treated with the anti-Thy-1.2 (1:5 of hybrid the tumoricidal assay. oma culture supernatant) followed by Low-Tox rabbit complement (1:20). Less than 0.5% of the remaining cells were Thy-1.2 positive by immunofluorescence. RESULTS Bone Marrow Derived Macrophages. Bone marrow cells from both femurs were used to generate monocyte colonies by the liquid culture Effect of CRP on Tumoricidal Activity of Inflammatory Mac methodology of Stewart (33). a-Minimal essential medium (Gibco, rophages. Inflammatory (elicited) peritoneal macrophages from Grand Island, NY) supplemented with L-glutamine (2.0 HIM),genta- C3H/HeN mice incubated with different concentrations of micin (50 Mg/ml), 10% FBS, 5% horse serum (Sterile Systems, Inc., purified CRP throughout a 48-h assay exhibited significant Logan, UT), 10% L-cell conditioned medium containing a high titer of tumoricidal activity against transformed target cells (Fig. I. !)â€¢ colony stimulating factor 1, and buffered with 0.01 M HEPES (pH 7.0) Maximum enhancement of tumoricidal activity occurred at 10- was used. After 10 days monocyte-macrophages were harvested by the /Â¿g/mldoses of CRP. Macrophages preincubated for 5 h with neutral protease Dispase II (Sigma) as described by others (34). CRP followed by washing prior to the addition of the target Macrophage Mediated Tumoricidal Activity. Tumoricidal activity of cells displayed a similar increase in their tumoricidal activity macrophages or macrophage cell lines was measured using a 24- or 48- h [3H]dThd release assay (35). Target cells at 1.5 x 10' cells in a 25- (Fig. IB). In all of these experiments, the background level of cm2 tissue culture flask were labeled with 100 Â¿iCiof low specific cytolytic activity of the untreated macropahges was minimal activity [3H]dThd (7.5 mCi/mmol; ICN, Irvine, CA) for 24 h for (<15%), indicating that the macrophages had not been activated adherence cells and 5-6 h for the nonadherent P815 cells. To assay for in vivo or by tissue culture components. Macrophages incubated iumeinculai activity the macrophage monolayers (2.5 x lOVwell) were with an optimal dose of MAF and LPS were included in all incubated with 0.2 ml of various dilutions of an activating agent and experiments to show that the macrophages could respond to washed with warm DMEM, after which 2.5 x IO4labeled target cells triggering signals (Fig. 1). The duration of exposure of macro were added to each well in 0.2 ml of DMEM (+10% FBS). Total phages to CRP that was necessary for the acquisition of tumor incorporated counts were determined by detergent (1% SDS) lysis of target cells. The amount of [3H]dThd released from 2.5 x IO4 target icidal activity was tested by simply exposing macrophage mon cells was: L-cells, 7-11 x IO3cpm; and CAKI-1, 3.5-7.0 x IO3 cpm. olayers to CRP for 0.25 to 9.0 h and then removing the CRP prior to the addition of the target cells. Tumoricidal activity Spontaneous release of label by the target cells alone and background release from the target cells incubated with nonstimulated macrophages greater than the background level was evident as early as 30 were measured. AH samples were run in triplicate. Results were calcu min after treatment with CRP and continued to increase up to lated as 2.0 h (Fig. 2). Since elicited macrophages are more responsive to LK (MAF) and LPS, we compared resident versus elicited % of specific release (% of cytoxicity) peritoneal macrophages for their responsiveness to CRP. Both _ Experimental release (cpm) - spontaneous release (cpm) cell populations showed a similar tumor cell cytotoxicity re x 100 Total cpm â€”spontaneous release (cpm) sponse pattern to different concentrations of CRP (Fig. 3). These results suggest that CRP could fully activate macro Statistical Analysis. The statistical significance of differences between phages in vitro to the level of expressing tumor cell killing. test groups was analyzed by Student's t test (two tailed). Since activated macrophages are known to selectively kill transformed tumor cell lines /'/; vitro but do not destroy non- Purification and Quantitation of CRP. CRP purification was per formed using the method outlined by Volanakis et al. (36). Briefly, transformed cells, we tested CRP-activated macrophages for pleural or ascites fluids from cancer patients were passed through a cytolytic activity against the P815 mastocytoma line, L-929 column of PC dipeptide conjugated agarose beads in the presence of murine fÃbroblasts,the human CAKI-1 carcinoma, and primary Ca2* (5 mM). The column was then washed extensively with TBS containing 5 mM ( Â¡i''. Bound CRP was eluted using 5 mM EDTA in explanted human fÃbroblasts.All three tumor targets were lysed; the human fÃbroblastswere not destroyed under identical con TBS. Fractions containing protein were pooled, recalcified, and allowed to bind to the affinity matrix again. After the column was washed with ditions (data not shown). TBS-Ca2*, the CRP was eluted by competing with 1 mM phosphoryl- Effect of CRP on Tumoricidal Activity of Mouse Macrophage choline (Sigma) (made with pyrogen free water) in TBS-Ca2+. The Cell Lines. Treatment of macrophage cell lines with purified protein was collected and dialyzed extensively against 0.15 M NaCl CRP resulted in a range of activation responses. The macro (0.02 M HEPES buffered) with 2 mM EDTA to dissociate bound phage cell lines, PU5 1.8, J774, and RAW 264.7, all acquired 5078

70 r A B

MAF + LPS Fig. 1. Tumoricidal activity of elicited mac rophages from C3H/HeN mice treated with purified human CRP. A. macrophages exposed to CRP throughout the 48-h assay period; B, macrophages exposed to CRP for S h prior to the addition of labeled CAKI-1 target cells. Effectortarget cell ratio was 10:1. A LK prep aration with MAF activity was used at a 1:64 dilution with LPS (10 ng/ml) as a triggering dose. Bars, SD.

0.05 0.10 1.0 3.0 10.0 30.0 100 1.0 10.0 30.0 100 CRP /Â¿g/ml CRP

TO 80 r

70 60

60 Â¿>50 'y MAF + LPS 'x O >, 50 o 40 o

O 40 SÂ« 30 30 20

IO O-

JL O 0.25 0.5 1.0 2.0 5.0 7.0 9.0 J Mrs of CRP Exposure 1.0 10.0 50.0 MAF+LPS Fig. 2. Effect of exposing macrophages to CRP for various intervals on the CRP /Â¿g/ml induction of tumoricidal capacity. Assay conditions are those described in Fig. 1. Fig. 4. Activation of mouse macrophage cell lines by purified human CRP. Bars, SD. Each macrophage cell line was exposed to different concentrations of CRP for 5.0 h prior to the addition of CAKI-I target cells at an effectortarget cell ratio of 10:1. Points, mean percentage of cytotoxicity of 3 experiments. Hun. SD.

cytotoxicity were observed in either the presence or absence of CRP (data not shown) and thus CRP does not enhance binding of A/J macrophages to the CAKI-1 or L-929 target cells. Role of LPS in CRP Mediated Activation. Since LPS is a very potent nonspecific activator of macrophages and a common contaminant of reagents, it was necessary to determine if acti vation was due to the presence of LPS in the CRP preparation. LPS also acts synergistically with LK in macrophage activation and therefore we tested if LPS was required for CRP-mediated activation. CRP containing less than 0.1 ng of LPS/ml by the Fig. 3. Comparison of the activation by CRP of resident versus elicited peritoneal macrophages from C3H/HeN mice. Macrophages exposed to CRP for Limulus assay activated inflammatory peritoneal macrophages 2.0 h before addition of CAKI-1 target cells at an effectortarget cell ratio of 10:1. from both C3H/HeN and C3H/HeJ mice to the same extent (Table 1). When the macrophages from these strains were the ability to kill tumor cells as demonstrated by the destruction incubated in the presence of a subactivating dose (1:64) of LK of L-929 mouse fibroblasts (Fig. 4). Optimal activation of PU5 (MAF) and a triggering dose (10 ng/ml) of LPS, elicited mac 1.8 and J774 cells required only 1 Â¿Â¿gofCRP/ml, whereas rophages from the C3H/HeN but not the C3H/HeJ strain RAW 264.7 cells required levels of 10 tig/ml The P388D, cell became activated and displayed significant tumoricidal activity. line was not significantly activated by CRP when compared to The same analysis was performed using the C57BL/10SN (LPS either the untreated controls or the inflammatory macrophages. responder) and C57BL/10ScN (LPS nonresponder) strain com Inflammatory (elicited) PEC from A/J mice contain macro binations as a source of macrophages with similar results (Table phages which are defective in their ability to bind tumor target 1). cells when assessed for activation (37). Only minimal levels of Polymyxin B irreversibly binds to LPS and neutralizes its 5079 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. CRP-MEDIATED MACROPHAGE ACTIVATION

Table 4 Effect of the specific removal ofCRP by either antibody or PC tors such as bacteria or their products, e.g., MDP (26), plant conjugated agarose beads on macrophage tumori cititiI activity lectins (41), and polyanions such as polyinosinic-polycytidylic Affinity purified goat IgG anti-human CRP or PC dipeptide was covalently linked to CNBr activated agarose beads and used to remove CRP from the culture acid or pyran copolymer (42, 43) are all exogenously derived. medium prior to testing for tumoricidal activity of inflammatory macrophages. Complement Factor B has recently been shown to activate Initial CRP % (v/v) Final CRP % of specific" macrophages and is the only example besides CRP of a nor Reagent on beads (jig/ml) of beads (/ig/ml) cytolysis mally occurring blood protein which is a direct macrophage Goat(IgG)PC anti-CRP Â±4.07.0 activator (44). Based on the findings of Deodhar et al. (29, 30) Â±6.020.0 Â±4.011.0 with liposome encapsulated CRP and the data presented here, CRP should be considered a nonimmunological, physiologically dipeptideNone101010100MAP Â±2.06.0 Â±5.023.0 relevant activator. CRP would be expected to be present at Â±1.021.0 inflamed sites in sufficient concentrations during the acute phase of a systemic inflammatory response to influence mac Â±1.019.0 Â±2.018.0 rophage activation. Â±2.525.5 The conclusion that CRP belongs in a distinct class of non- Â±3.06.0 Â±1.033.5 immune macrophage activators is based on several direct lines + LPS501015010150101None50None2.01.06.01.00.57.09.08.09.59.5009.0Â±3.0 of evidence from these experiments, (a) Our human CRP prep " Tumoricidal assay was that described in Table 1. Mean Â±SD of two identical arations were purified to the extent that they were free of experiments. detectable levels of other blood proteins using immunological assays (ELISA) that readily detect ng/ml amounts. A well characterized macrophage activator such as 7-interferon could 60 possibly copurify with CRP. However, CRP prepared by affinity chromatography is devoid of any antiviral activity and the -y-

50 interferon level detected by radioimmunoassay is <0.5 unit/ ml.3 The possibility that activation was due to a low molecular -CRP weight component bound tightly to CRP was not ruled out by u these experiments, (b) Activation did not require the presence Â£30 of a lymphocyte product (LK) or a second triggering signal of o LPS. Removal of any remaining adherent T-cells present LU among the PEC failed to alter the responsisveness of the -CRP macrophages to the CRP. (c) Selective removal of CRP from IO the assay medium either with antibody or by a solid phase ligand (PC) for CRP also abolished the macrophage activating component. Although these procedures do not formally exclude 16 32 64 Â¡28 the possibility that the CRP molecule might bind a low molec 1/LK (MAP) ular weight substance that lowers the threshold of macrophages Fig. 6. Effect of CRP (10 fig/ml) on the macrophage tumoricidal activity to other triggering signals, they strongly suggest that activation induced by different concentrations of IK (MAM CRP alone at 10 fig/ml resulted in 40.5 Â±4.4% specific cytolysis. Elicited macrophages from C3H/HeN mice is mediated by CRP. were incubated throughout the 48-h assay with the Ik (MAP) and CRP. A, Because LPS is a very potent activator of macrophages, the cytolysis in the absence of CRP or LK. issue of whether it was a significant component in our test system was carefully examined. The concentration of LPS in increased tumoricidal activity at LK (MAF) concentrations that our reagents was lower than the level needed to directly trigger were less than optimal (Fig. 6). The increase in cytotoxicity in macrophages (27) and was also lower than the concentration the presence of both activators was additive rather than syner- (1.0 to 10.0 ng/ml) used by others as a triggering dose for LK gistic. The data shown in Fig. 6 was obtained with C3H/HeN activated cells (38). Removal of LPS or neutralization of its macrophages; however, the same enhancement by CRP in the activity with polymyxin B did not alter activation by CRP. presence of small amounts of LK (MAF) was also observed Furthermore, macrophages from two LPS nonresponder strains with C57BL/6 macrophages (40). were activated by CRP to the same extent as cells from the syngeneic LPS responder strains. CRP is a ligand with binding DISCUSSION sites for PC and polycations but does not bind to LPS (45). Therefore, we conclude that LPS is not likely to contribute to Macrophage effector functions are the result of the response the activation mediated by human CRP. of cells of the monocytic lineage to stimuli derived from both Since inflammatory and immune events may occur simulta specific immune responses and nonspecific inflammatory re neously, the interaction of the products of inflammatory (CRP) sponses. One such effector function extensively examined in and immune (â€¢y-interferonorLK) responses at the level of the vitro is the activation of the macrophage to a level that is readily macrophage might be expected. Our results show that although quantitated by the selective cytolysis of neoplastic cells. Using LK activation usually resulted in more cytolytic activity than quite different macrophage-tumor target cell combinations, sev treatment with CRP, exposure of cells to both reagents over a eral investigators have established that elicited macrophages range of LK concentration did not reveal any synergism or become cytolytic in response to LK preparations via a two-step inhibition of tumoricidal activity. Purified recombinant 7-inter process that involves priming by MAF followed by a triggering signal provided either by a 1-10-ng/ml dose of LPS or by using feron is currently being tested in combination with purified CRP to determine whether CRP can influence LK dependent higher initial concentrations of MAF (38). LPS at higher con activation. It is conceivable that if other non-T-cell products centrations than the triggering dose is sufficient by itself to activate macrophages in the absence of LK (27). Other activa 3Unpublished observation. 5081 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1986 American Association for Cancer Research. CRP-MEDIATED MACROPHAGE ACTIVATION

7. Pepys, M. B., and Baltz, M. L. Acute phase proteins with special reference activate macrophages, the mechanism will involve distinct re to C-reactive protein and related proteins (Pentaxins) and serum amyloid A ceptors. protein. Adv. Immunol., 34:141-212, 1983. Although the CRP used in these studies was isolated from 8. Kushner, I., and Kaplan, M. H. Studies of acute phase protein. I. An human fluids, it was tested in a mouse macrophage tumoricidal immunohistochemical method for the localization of C-reactive protein in rabbits. Association with necrosis in local inflammatory lesions. J. Exp. system since the mouse system has been extensively character Med., 114:961-975, 1961. ized. Furthermore, there is considerable controversy as to 9. Du Clos, T. W., Mold, C., Paterson, P. Y., Alroy, J., and GewÃ¼rz,H. Localization of C-reactive protein in inflammatory lessions of experimental whether human monocytes or NK cells are responsible for the allergic encephalomyelitis. Clin. Exp. Immunol., 43:565-571, 1981. in vitro killing measured in assay systems for human monocytes 10. Osmand, A. P., Friedenson, B., GewÃ¼rz,H.,Painter, R. H., Hofman, T., and (46). Mice have trace levels of CRP estimated at 100-200 ng/ Shelton, E. Characterization of C-reactive protein and the complement component Clt as homologous proteins displaying cyclic Dentamene sym ml that change slightly during inflammation (7) and therefore metry (pentraxins). Proc. Nati. Acad. Sci. USA, 74: 739-743, 1977. the effects on macrophages of endogenous levels of CRP would 11. Osmand, A. P., Gerwurz, H., and Friedenson, B. Partial amino acid sequences of human C-reactive proteins: homology with immunoglobulins and histo- be minimal. compatibility antigens. Proc. Nati. Acad. Sci. USA, 74:1214-1217, 1977. The mechanism whereby CRP activates macrophages is 12. Oliveira, E. B., Gotschlich, E. C., and Liu. T-Y. Primary structure of human largely unknown. Investigations by Deodhar et al. (30) showed C-reactive protein. J. Biol. Chem., 254:489-502,1979. 13. While-head, A. S., Bruns, G. A. P., Markham, A. F., Colten, H. R., and increased Superoxide aniÃ³nproduction by macrophages from Woods, D. E. Isolation of human C-reactive protein cDNA and localization mice treated with both liposome encapsulated CRP and free of the gene to chromosome 1. Science (Wash. DC), 221:69-71, 1983. CRP, although the former was 10 to 100 times more effective. 14. Volanakis, J. E., and Kaplan, M. H. Specificity of C-reactive protein for choline phosphate residues on pneumococcal C-polysaccharide. Proc. Soc. Studies under way in this laboratory indicate that CRP binds Exp. Biol. Med., 136:612-616, 1971. to a subset of 30-35% of the peroxidase positive macrophages 15. DeCameli, R., Potempa, L. A., Siegel, J., Suyehira, L., Petras, K., and GewÃ¼rz,H. Binding reactivity of C-reactive protein for polycations. J. that infÃltrateas.c. inflammatory site. The binding appears to Immunol., 125: 1933-1949, 1980. involve high affinity receptors, perhaps Fc receptors. CRP has 16. Mortensen, R. F., Osmand, A. P., Lint, T. F., and GewÃ¼rz,H.Interaction of been reported to bind to human lymphocyte populations with C-reactive protein with lymhocytes and monocytes: complement-dependent adherence and phagocytosis. J. Immunol., 117: 774-779, 1976. Fc receptors (22) and has been shown to be opsonic through 17. Mortensen, R. F., and Duszkiewicz, J. Mediation of CRP-dependent phag macrophage Fc receptors (17). Binding of monomeric CRP to ocytosis through mouse macrophage Fc receptors. J. Immunol., 119: 1611- both human monocytes (23,48) and mouse monocytes (47) has 1616, 1977. 18. Kilpatric, J. M., and Volanakis, J. E. Opsonic properties of C-reactive been reported previously. CRP determinants have been detected protein. Stimulation by phorbol myristate acetate enables human neutrophils on human NK cells and may be involved in the lytic function to phagocytose C-reactive protein-coated cells. J. Immunol., 134: 3364- 3370, 1985. of these effectors (49). 19. Kaplan, M. H., and Volankis, J. E. Interaction of C-reactive protein com Since CRP is known to be deposited at sites of tissue destruc plexes with the complement system. I. Consumption of human complement tion (8, 9), it might be expected to exert its influence on associated with the reaction of C-reactive protein with pneumococcal C- polysaccharide and with choline phosphatides, lecithin and sphingomyelin. macrophages infiltrating these sites. The local environment has J. Immunol., 112:2135-2147, 1974. been proposed as an important modulator of macrophage effec 20. Claus, D. R., Siegel, J., Petras, K., Osmand, A. P., and GewÃ¼rz,H. Interac tions of C-reactive protein with the first component of human complement. tor functions; however, the activity (tumoricidal) measured was J. Immunol., 119:187-196, 1977. inhibited (50). CRP may be a significant component of a 21. Mold, C., and GewÃ¼rz,H. Inhibitory effect of C-reactive protein on alterna localized inflammatory site and would serve as a positive mod tive C pathway activation by liposomes and Streptococcus pneumonia. J. Immunol., 127: 2089-2092, 1981. ifier in an environment where infiltrating macrophages exert 22. James, K.. Hansen, B. and GewÃ¼rz,H. Binding of C-reactive protein to their effector activities. Since CRP was originally detected by human lymphocytes. II. Interaction with a subset of cells bearing the Fc relatively insensitive immunofluorescent methods at the site of receptor. J. Immunol., 127: 2546-2550, 1981. 23. Whisler, R., Newhouse, Y., and Mortensen, R. F. 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5083

Kamyar Zahedi and Richard F. Mortensen

Cancer Res 1986;46:5077-5083.

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