Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14429–14434, November 1998 Medical Sciences

Fetuin (␣2-HS-glycoprotein) opsonizes cationic macrophage- deactivating molecules (CNI-1493͞p38 mitogen-activated kinase)

HAICHAO WANG*†‡,MINGHUANG ZHANG†,MARINA BIANCHI†,BARBARA SHERRY†,ANDREW SAMA*, AND KEVIN J. TRACEY†§

Departments of *Emergency Medicine and §Surgery, North Shore University Hospital-New York University School of Medicine, and †The Picower Institute for Medical Research, Manhasset, NY 11030

Communicated by George J. Todaro, University of Washington, Seattle, WA, September 22, 1998 (received for review February 5, 1998)

ABSTRACT Macrophages become activated by bacterial of circulating fetuin, but it also is expressed by mono- endotoxin (lipopolysaccharide) and other stimuli to release cyte͞macrophages (26). The fetuin promoter region has several proinflammatory cytokines and NO. To prevent release of toxic potential interleukin 6-responsive elements (27), and its synthesis or potentially lethal quantities of these factors, the state of is down-regulated during injury and inflammation (20). Fetuin is macrophage activation is counter-regulated by anti-inflamma- an acidic glycoprotein with three N-linked and three O-linked tory mediators (e.g., glucocorticoid hormones, interleukin 10, oligosaccharide chains, whose terminal sugar residues are rich in ␤ and transforming growth factor type ). Fetuin, a negative sialic acid (N-acetylneuraminic acid), contributing to its net acute-phase protein, recently was implicated as an anti- negative charge (28). A role for fetuin as a carrier of bioactive inflammatory mediator, because it is required for macrophage molecules has been proposed based on observations that it binds deactivation by spermine. In the present studies, we found that and carries Ca2ϩ ion (22). fetuin is necessary for macrophages to respond to CNI-1493, a We recently proposed that fetuin occupies an important role in tetravalent guanylhydrazone inhibitor of p38 mitogen-activated counter-regulating macrophage activation (29). This hypothesis protein kinase phosphorylation. Fetuin dose-dependently in- was based on our observations that spermine, a ubiquitous creases macrophage uptake of CNI-1493, which can be specifi- biogenic polyamine that accumulates at sites of injury or inflam- cally inhibited by anti- fetuin antibodies. Anti-human fetuin antibodies render primary human peripheral blood mono- mation, inhibits macrophage cytokine synthesis only in the pres- nuclear cells insensitive to deactivation by CNI-1493. Thus, ence of fetuin (29). Thus, macrophages use fetuin to assess the macrophages use fetuin as an opsonin for cationic-deactivating abundance of extracellular spermine, which, in turn, down- molecules, both endogenous (e.g., spermine) and pharmacologic regulates synthesis of proinflammatory cytokines and prevents (e.g., CNI-1493). This role of fetuin as an opsonic participant in excessive inflammation (11, 29). It previously was unknown macrophage-deactivating mechanisms has implications for un- whether the interaction of macrophages with fetuin (and thereby derstanding and manipulating the innate immune response. any fetuin-associated small molecules) might be integral to the state of macrophage activation. CNI-1493 is a tetravalent gua- The pathophysiological sequelae of infection, trauma, and isch- nylhydrazone compound that prevents phosphorylation of p38 emia are mediated by macrophage-derived proinflammatory mitogen-activated protein (MAP) kinase and inhibits macro- cytokines. Regulation of macrophage cytokine responses is tightly phage activation (30–33). Here we demonstrate that fetuin binds controlled, because a relative overproduction of these mediators CNI-1493 and specifically enhances cellular uptake by macro- can be detrimental to organ homeostasis and survival (1, 2). To phages. Thus, fetuin mediates macrophage deactivation by opso- prevent such morbid sequelae, a number of endogenous counter- nizing both endogenous and therapeutically administered cat- regulating mechanisms have evolved to suppress macrophage ionic cytokine synthesis inhibitors that restrain the innate immune activation, including the release of glucocorticoid hormones (3), response. anti-inflammatory cytokines [transforming growth factor type ␤ and interleukin (IL) 4, IL-10, IL-11, and IL-13] (4–8), prosta- MATERIALS AND METHODS glandin E2 (9), and spermine (10, 11). Hepatic-derived acute- Cell Culture. Murine macrophage-like RAW 264.7 cells were phase (APP) whose levels rise (positive APP) or fall obtained from the American Type Culture Collection and cul- (negative APP) during acute inflammation or injury also can tured in RPMI medium 1640 (GIBCO͞BRL) supplemented participate in the regulation of macrophage activities (12–14). with 10% fetal bovine serum (FBS) and 0.2 mM glutamine. These counter-regulatory mediators occupy an important func- Cultures were maintained at 37°C in a humidified atmosphere of tion in deactivating macrophages and restraining the innate 5% CO2 and 95% air. At 80–90% confluency, RAW 264.7 cells immune response. were harvested by gentle scraping, resuspended at 106 cells͞ml in ␣ Fetuin and its human homologue ( 2-HS-glycoprotein) are RPMI medium 1640͞10% FBS͞0.2 mM glutamine, seeded onto negative acute-phase proteins (15–17); normal circulating levels 6 ͞ ␮ ͞ 24-well tissue culture plates (10 cell well), and precultured for in adults (300–600 g ml) fall significantly (30–50%) during 2 hr to allow cell adherence. Human peripheral blood monocytes injury and infection (18–20). The biological role of fetuin is (HuPBMCs) were isolated by density gradient centrifugation unknown, although it has been implicated as an immunomodu- through Ficoll (Ficoll-Paque PLUS, Pharmacia), and resus- lator that can participate in stimulation of bacterial phagocytosis pended in RPMI medium 1640 supplemented with 10% heat- by neutrophils (21) and promotion of endocytosis by mouse inactivated human serum͞0.2 mM L-glutamine as described (11). macrophages (22–25). Hepatocytes are the principal cell source After incubation overnight at 37°C, nonadherent cells were

The publication costs of this article were defrayed in part by page charge Abbreviations: HuPBMC, human peripheral blood mononuclear cell; payment. This article must therefore be hereby marked ‘‘advertisement’’ in LPS, lipopolysaccharide; TNF, tumor necrosis factor; FBS, fetal accordance with 18 U.S.C. §1734 solely to indicate this fact. bovine serum; MAP, mitogen-activated protein. © 1998 by The National Academy of Sciences 0027-8424͞98͞9514429-6$2.00͞0 ‡To whom reprint requests should be addressed. e-mail: hwang@ PNAS is available online at www.pnas.org. picower.edu.

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removed, and adherent monocyte-enriched cultures then were pooled, boiled for 3 min in SDS sample buffer, and resolved on stimulated with lipopolysaccharide (LPS). 10% SDS͞PAGE gels at constant current (25 mA). After elec- LPS Stimulation and Bioassay of CNI-1493-Enhancing Activ- trophoresis, the gel was stained briefly (for 5 min) with Coomas- ity. CNI-1493 was added either alone or in combination with sie blue (1.25% Coomassie blue R250 in 30% methanol͞10% macrophage proteins to RAW cell or HuPBMC cultures that had acetic acid), and then destained in 30% methanol͞7% acetic acid. been preincubated for various times in media containing specific After destaining, the protein band corresponding to the CNI- concentrations of added serum. One hour later, freshly sonicated 1493-enhancing activity was excised from the SDS͞PAGE gel, Escherichia coli endotoxin LPS (100 ng͞ml, Sigma) was added. washed thoroughly with water, and submitted for N-terminal Four hours after LPS stimulation, culture supernatants were amino acid sequencing (Commonwealth Biotechnologies, Rich- assayed for tumor necrosis factor (TNF) by ELISA using mono- mond, VA). clonal or polyclonal antibodies against murine or human TNF as Effects of Fetuin on CNI-1493-Mediated Suppression of TNF described (11). The CNI-1493-enhancing activity was defined as Release. To confirm the role of fetuin in enhancing the suppres- Ϫ [% inhibition of TNF release(CNI-1493 ϩ macrophage fraction)] [% sion of TNF release from activated macrophages by CNI-1493, ␣ inhibition of TNF release(CNI-1493 alone)], where one arbitrary unit purified bovine fetuin (F3004, Sigma), human 2-HS-glycopro- was defined as the amount of macrophage fraction that produces tein (G0516, Sigma), or antiserum specific for human fetuin was a 10% further suppression of TNF release in the presence of added cocurrently with CNI-1493 to RAW 264.7 or primary CNI-1493. HuPBMC cultures. Note that fetuin was purified and rendered Western Blotting Analysis of p38 MAP Kinase. Thirty minutes endotoxin free before use in any experiments. Polyclonal anti- ͞ ␣ after stimulation with LPS (100 ng ml) in the absence or bodies against purified 2-HS-glycoprotein were generated in presence of either CNI-1493, SB203580, and͞or fetuin, RAW rabbits, and specificity was confirmed both by ELISA and West- 264.7 cells were lysed immediately in SDS sample buffer (62.5 ern blotting analysis using purified human fetuin following stan- mM Tris⅐HCl, pH 6.8͞2% SDS͞10% glycerol͞50 mM dard protocols. To examine the effect of fetuin in vivo, CNI-1493 DTT͞0.1% bromphenol blue). The concentration of phospho- was injected i.p. into BALB͞c mice either alone or in combina- p38 MAP kinase was measured by Western blotting analysis using tion with bovine fetuin 1 hr before LPS treatment. Two hours the PhosphoPlus p38 MAP Kinase Antibody Kit following the after LPS stimulation, mice were bled, serum was collected, and manufacturer’s instructions (New England Biolabs). To verify serum TNF levels were determined by TNF ELISA. equal loading for different samples, the signal for phospho-p38 Interaction of Fetuin with CNI-1493. To examine whether the MAP kinase was stripped, and the membrane was reprobed with acidic glycoprotein fetuin binds to the cationic CNI-1493 mole- a different antibody specific for total p38 MAP kinase as in- cule, CNI-1493 (10–100 ␮M) was mixed with different concen- structed by the manufacturer (New England Biolabs). trations of fetuin (0–25 ␮M) and incubated at room temperature Fractionation of RAW 264.7 Protein by Ultrafiltration. RAW for 30 min, and the mixture was fractionated by ultrafiltration 264.7 cell cultures at 80–90% confluency were washed extensively using Amicon Centricon-10. Unbound CNI-1493 in the filtrate with, and then cultured in, OPTI-MEM I medium fraction was reacted with the Urea Nitrogen reagents (BUN acid (GIBCO͞BRL). At different time points, the RAW 264.7 cells reagent:BUN color reagent ϭ 3:2, Sigma), and the concentration and conditioned serum-free medium were collected separately. of unbound CNI-1493 in the filtrate fraction was calculated by Cells were lysed by three freeze-thaw cycles and fractionated by comparison with OD570 of standard solutions of CNI-1493. To successive ultrafiltration through Amicon membranes with Mr investigate whether fetuin affected the binding or uptake of cutoff ranges of 100 kDa and 30 kDa, respectively (Amicon). For CNI-1493 to macrophages, fetuin (1–100 ␮g͞ml) was added ion-exchange chromatography, 2–3 liters of the pooled RAW concurrently with radio-labeled (14C)-CNI-1493 (1–5 ␮M) to 264.7-conditioned medium were clarified by filtration through serum-deprived RAW 264.7 cultures, and incubated for 30 min 0.2-␮M filters, and proteins in the medium were concentrated either at 4°C or 37°C. After three extensive washes with 1ϫ PBS, under N2 at 4°C by ultrafiltration on an Amicon apparatus with cells were lysed for 10 min with 1 N NaOH, cell lysates were YM-100 (Mr cutoff of 100 kDa) and YM-30 membranes (Mr transferred to a 96-well Luma Plate 96, and radioactivity was cutoff of 30 kDa). measured by using a microplate scintillation counter (Packard). Mono-Q Chromatography. The 30- to 100-kDa protein frac- Statistical Analysis. All values in the figures and text are tion of the RAW 264.7-conditioned medium was loaded onto a expressed as mean Ϯ SEM of 2–3 independent experiments with 1-ml Mono-Q column (Mono-Q HR5͞5, Pharmacia) pre- each treatment in triplicate or quadruplicate. Student’s two- equilibrated in buffer B (50 mM Tris⅐HCl, pH 7.5͞150 mM sample t test was used to compare means between groups. NaCl), and the column was washed with buffer B at 0.5 ml͞min until the A280 dropped below 1% of its maximum. Proteins bound RESULTS to the column were eluted in 1-ml fractions over 25 min with a Identification of a Single Protein Required for Macrophage linear gradient of NaCl in buffer B increasing from 150 mM to 2 Response to CNI-1493. Previous studies demonstrated that CNI- M. Fractions were concentrated by Amicon Centricon-10, and 1493 suppresses the production of TNF from LPS-activated ␮ aliquots were assayed for activity and polypeptide migration on macrophage cultures with an IC50 of approximately 1.5 M (30, SDS͞PAGE. 31). When RAW 264.7 cells were cultured (in RPMI medium SDS͞PAGE. Mono-Q fractions were mixed with 1 vol of 1640͞10% FBS͞0.2 mM glutamine) for Ͼ56 hr, however, the sample solubilization buffer (2% SDS͞10% ␤-mercaptetha- level of LPS-stimulated TNF production was not reduced by nol͞0.03% bromophenol blue͞1.25 M Tris⅐HCl, pH 7.0), boiled CNI-1493, even at concentrations of up to 10 ␮M (Fig. 1). for 5 min, and resolved on 4–20% gradient SDS͞PAGE gels Macrophage cultures that failed to respond to CNI-1493 were (Bio-Rad). After electrophoresis, the gel was silver-stained by termed CNI-1493-unresponsive. Interestingly, these CNI-1493- using the Silver Plus kit (Bio-Rad). To determine the molecular unresponsive cells maintained their responsiveness to a selective weight of the proteins contributing to CNI-1493-enhancing ac- inhibitor of p38 MAP kinase (SB203580), as judged by effective tivity, the gel was sliced into 2-mm segments that were crushed suppression of LPS-induced phosphorylation of p38 MAP kinase ϫ individually into small fragments in 1 PBS. After gentle shaking (data not shown), as well as inhibition of NO production (IC50 for ϭ ␮ at room temperature overnight, gel fragments were removed by CNI-1493-responsive cultures 5–7.5 M, vs. IC50 for CNI-1493- centrifugation and proteins in the supernatant were concentrated unresponsive ϭ 6–8.5 ␮M). CNI-1493-unresponsive cultures by ultrafiltration with Centricon-10 and assayed for CNI-1493- were not LPS tolerant, because LPS stimulation caused maximal enhancing activity. production of TNF and NO. We therefore assessed whether a N-Terminal Amino Acid Sequencing. Mono-Q fractions con- macrophage cell-associated factor could restore the responsive- taining the major peak of CNI-1493-enhancing activity were ness of CNI-1493-unresponsive macrophage cultures. Normally Downloaded by guest on September 28, 2021 Medical Sciences: Wang et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14431

2%). Addition of the 30- to 100-kDa fraction did not affect TNF release in the absence of CNI-1493, indicating that both CNI-1493 and a 30- to 100-kDa macrophage-associated factor were re- quired to suppress macrophage TNF production. Protein Purification and Identification as Fetuin. The respon- sible factor was purified by fractionating the 30- to 100-kDa fraction of the RAW 264.7-conditioned medium via anion ex- change chromatography. More than 65% of the 30- to 100-kDa fraction (by mass) had a neutral or net positive charge at neutral pH, did not bind to the Mono-Q column, and was removed by washing with buffer containing 150 mM NaCl. These flow- through fractions from the anion exchange resin did not restore the responsiveness of serum-depleted macrophage cultures to CNI-1493, as the level of TNF was not changed when added concurrently with CNI-1493 (data not shown). Proteins retained on the Mono-Q column were eluted by a linear gradient of NaCl from 0.15 to 2.0 M, and two peaks of CNI-1493-enhancing activity FIG. 1. Identification of CNI-1493-unresponsive cells. RAW 264.7 cell cultures were exposed to CNI-1493 1 hr before LPS stimulation, and were detected: a minor peak in a low salt (0.25–0.5 M NaCl) fraction and a major peak in higher salt (1.7–1.9 M NaCl) TNF levels in the supernatant were determined by ELISA 4 hr after LPS ͞ treatment. All data are expressed as % of control (ϩ LPS alone). For fractions. SDS PAGE gel analysis of the Mono-Q fractions RAW 264.7 cells precultured under normal conditions, CNI-1493 pro- containing the major peak of activity revealed two prominent ᮀ Ϸ Ϸ moted a dose-dependent suppression of TNF release ( ), with an polypeptide bands migrating at the Mr 65–67 and 55–56 kDa, ␮ estimated IC50 of 1.5 M. When RAW 264.7 cells were precultured under along with a small number of faint bands of lower molecular serum-deprived conditions, they became unresponsive to CNI-1493 (E). masses (15–30 kDa, Fig. 2A). To determine which protein(s) contributed to the CNI-1493-enhancing activity, the SDS͞PAGE cultured RAW 264.7 cells cellular proteins were fractionated by gel was cut into 2-mm slices, the proteins were eluted by incu- ultrafiltration and fractions were assayed for an activity that Ͼ bation in PBS, and activity was determined by addition of eluted rendered cells responsive to CNI-1493. The majority ( 90%) of proteins to unresponsive macrophage cultures. The CNI-1493- Ͼ partially fractionated whole-cell proteins ( 100 kDa) did not enhancing activity localized to the gel slice containing a 65–67 restore macrophage response to CNI-1493, as evidenced by the kDa protein (Fig. 2B). The N-terminal amino acid sequence of continued production of TNF after LPS stimulation in the the bioactive protein was: XPLDPVAGYKEPAXDXXXTE- presence of CNI-1493. The 30- to 100-kDa fraction substantially QAALA. Comparison of this N-terminal amino acid sequence reduced TNF production in the presence of 1.5 ␮M CNI-1493 with the protein sequence database (PIR) revealed 100% identity (50 Ϯ 12%) as compared with controls (ϩ CNI-1493 alone, 4 Ϯ to bovine fetuin.

FIG. 2. Determination by preparative SDS͞PAGE gel elution of the protein responsible for CNI-1493-enhancing activity. (A) SDS͞PAGE. The Mono-Q fractions containing the major CNI-1493-enhancing activity were pooled, fractioned on a 4–20% gradient polyacrylamide gel, and stained with silver. Two major bands with molecular masses of 65–67 and 55–56 kDa along with several minor bands in the range of 15–30 kDa were detected. (B) SDS͞PAGE gel elution. Duplicate lanes of the preparative SDS͞PAGE gel were sliced (at 2 mm in width). Proteins in each slice were eluted into 1 ϫ PBS and added concurrently with CNI-1493 in the LPS challenge assay. CNI-1493-enhancing activity was eluted from only one slice, containing the prominent 65-to 67-kDa protein band. Downloaded by guest on September 28, 2021 14432 Medical Sciences: Wang et al. Proc. Natl. Acad. Sci. USA 95 (1998)

centration of CNI-1493 (2.5 ␮M) promoted a more than 50 Ϯ 12% suppression of TNF release (P ϭ 0.007). Bovine fetuin alone did not affect TNF release from LPS-stimulated macrophage cultures (data not shown). The terminal sialic acid residues on the oligosaccharide chains of bovine fetuin are required to mediate macrophage responsiveness to CNI-1493, because similar con- centrations of asialofetuin (sialic acid residues of the fetuin removed by neuraminidase) and CNI-1493 (2.5 ␮M) failed to restore the suppression of TNF release (TNF ϭ 11 Ϯ 7%, P Ͼ 0.05). Several other glycoproteins (e.g., glycophorin, ␣1-acid glycoprotein, human glycoprotein, and albumin) also were tested for CNI-1493-enhancing activity, but none restored macrophage responsiveness to CNI-1493, confirming that the observed CNI- 1493-enhancing activity is specific to fetuin. When considered together, these observations give evidence that macrophage cultures adopt fetuin from FBS in the culture medium and that fetuin is required for macrophage deactivation by CNI-1493. FIG. 3. Effect of fetuin on binding and uptake of CNI-1493 to Fetuin Increases CNI-1493 Uptake to CNI-1493-Unresponsive macrophages. RAW 264.7 cells were precultured under serum-deprived Cells. To investigate the mechanism by which fetuin restores the conditions until confluency, resuspended into fresh RPMI medium macrophage responsiveness to CNI-1493, we first tested whether 1640͞10% FBS, and plated on 96-well tissue culture plate at 5 ϫ 105 fetuin binds CNI-1493. When mixtures of fetuin and CNI-1493 ͞ cells well per 0.2 ml. After incubation at 37°C for 2 hr to allow adherence, were fractionated by ultrafiltration, we observed a dose- 14 radiolabeled C-CNI-1493 was added to the cultures either alone or in dependent decrease of CNI-1493 levels in the filtrate (Ͻ10 kDa) the presence of increasing concentrations of fetuin (0–50 ␮g͞ml) and incubated for 30 min at 4°C or 37°C. After three extensive washings, the fraction, indicating binding of CNI-1493 to fetuin. Based on the cell-associated radioactivity (cpm͞well per 5 ϫ 106 cells) was measured decrease of CNI-1493 concentration in the filtrate fraction, it is and expressed as mean Ϯ SEM (n ϭ 4). estimated that 1 ␮mol of fetuin binds 3–4 ␮mol CNI-1493. Asialofetuin, however, showed significantly less binding (Ͻ10%) Confirmation of the Role of Fetuin. To confirm that fetuin of CNI-1493 under the same conditions, indicating that efficient restores the responsiveness of macrophages to CNI-1493, puri- binding depends on intact sialic acid groups. Macrophage binding fied, endotoxin-free bovine fetuin was added in combination with of CNI-1493 was not significantly affected by bovine fetuin when CNI-1493 to unresponsive RAW 264.7 cell cultures. When added measured at 4°C, but at 37°C, fetuin dose-dependently increased alone, CNI-1493 (2.5 ␮M) caused a less than 5 Ϯ 5% suppression (by as much as 4- to 5-fold) the cellular uptake of CNI-1493 in of TNF release from LPS-stimulated RAW cell cultures. In the unresponsive macrophages (Fig. 3). Thus, fetuin binds CNI-1493 presence of bovine fetuin (10 ␮g͞ml), however, the same con- and specifically facilitates uptake into macrophages.

FIG. 4. Fetuin restored the suppressive effect of CNI-1493 on LPS-induced phosphorylation of p38 MAP kinase. Normally cultured (responsive cells, A), or serum-deprived (nonresponsive cells, B and C) RAW 264.7 cells were pretreated with CNI-1493 either alone (A and B) or in combination with fetuin (C) 1 hr before LPS stimulation (100 ng͞ml). Thirty minutes after LPS stimulation, cells were lysed and assayed for concentration of phosphorylated p38 by Western blotting. After detection of the signal for phospho-p38 MAP kinase (P Ϸ p38), the same membranes were stripped and reprobed with antibodies against the total p38 MAP kinase (supplied in the kit, total p38) to verify equal loading of different samples. Downloaded by guest on September 28, 2021 Medical Sciences: Wang et al. Proc. Natl. Acad. Sci. USA 95 (1998) 14433

FIG. 5. Effect of human fetuin (␣2-HS-glycoprotein) on CNI-1493-mediated suppression of TNF production. (A) Effect of human fetuin (␣2-HS-GP) on CNI-1493-mediated suppression of HuPBMCs. Human ␣2-HS-glycoprotein was added concurrently with 0.5 ␮M CNI-1493. Four hours after LPS stimulation, TNF levels in the supernatants were measured and expressed as % of control (ϩ LPS alone). (B) Effect of ␣2-HS-glycoprotein-specific antibodies on suppression of HuPBMCs by CNI-1493. Polyclonal antiserum against human ␣2-HS-glycoprotein was added (5.0 or 50 ␮l͞ml) with CNI-1493 (1.0 ␮M) 1 hr before LPS stimulation. Note that human fetuin-specific antiserum abrogated the suppression of HuPBMCs by CNI-1493.

Effect of Fetuin on CNI-1493-Mediated Suppression of p38 indicate that macrophage-associated fetuin opsonizes CNI-1493, MAP Kinase Phosphorylation. Because the deactivating mech- which prevents activation of p38 MAP kinase signaling. anism of action for CNI-1493 in macrophages depends on inhib- Effect of Human Fetuin on the Suppression of HuPBMCs by iting the phosphorylation of p38 MAP kinase, we next examined CNI-1493. We wanted to confirm that these observations were the effect of fetuin on this signaling pathway. As shown in Fig. 4A, not an artifact of murine macrophage cell lines cultured with and in agreement with previous work (33), CNI-1493 dose- bovine serum, and accordingly examined whether human fetuin ␣ dependently suppresses the LPS-induced phosphorylation of p38 ( 2-HS-glycoprotein) enhances the CNI-1493-mediated suppres- MAP kinase. When macrophages were depleted of fetuin by sion of TNF production from LPS-stimulated HuPBMCs cul- preincubation under serum-deprived conditions, CNI-1493 failed tured in human serum. As shown in Fig. 5A, CNI-1493 alone (500 to block the LPS-induced phosphorylation of p38 (Fig. 4B). A nM) caused Ͻ22% suppression of TNF release (P Ͼ 0.05), ␣ ␮ ͞ structurally unrelated pyridinyl imidazole inhibitor of p38 activity whereas coaddition of 2-HS-glycoprotein (10 g ml) resulted in (SB203580) effectively inhibited p38 in these serum-deprived significantly greater suppression of TNF release (Ͼ68 Ϯ 9%, P ϭ macrophage cultures, suggesting that fetuin is not required di- 0.007). Furthermore, when polyclonal antiserum against human ␣ rectly to inhibit p38 activity. When coadded with CNI-1493, fetuin 2-HS-glycoprotein was added to normally cultured HuPBMCs, dose-dependently restored the suppressive effect of CNI-1493 on the CNI-1493-mediated TNF suppression was significantly abro- the phosphorylation of p38 (Fig. 4C). When added alone, fetuin gated (Fig. 5B). Control (preimmune) serum did not affect the did not affect phosphorylation of p38 MAP kinase (Fig. 4C). suppressive effect of CNI-1493, indicating that HuPBMCs spe- Notably, no difference was found in the basal status of p38 cifically use fetuin to become deactivated by CNI-1493. phosphorylation (in the absence of LPS) between the CNI-1493- CNI-1493-Enhancing Activity in Vivo. To exclude the unlikely responsive and -unresponsive macrophage cultures (Fig. 4). possibility that the observed effects were limited to in vitro culture Moreover, when stimulated with equal concentrations of LPS conditions, we also tested whether fetuin enhanced the thera- (100 ng͞ml, for 30 min), no difference was found in the amplitude peutic activity of CNI-1493 in vivo. Bovine fetuin was adminis- of p38 phosphorylation. When considered together, these results tered i.p. to mice in conjunction with CNI-1493 1 hr before LPS

FIG. 6. Fetuin increased the CNI-1493-mediated suppression of serum TNF level in vivo. Fetuin (20 mg͞kg) was coadministered with CNI-1493 (2.5 mg͞kg) i.p. into BALB͞c mice 1 hr before LPS (10 mg͞kg) stimulation. Two hours after LPS stimulation, mice serum was collected and TNF levels were assayed by ELISA. Data shown are mean Ϯ SEM of two independent experiments with five per group. Downloaded by guest on September 28, 2021 14434 Medical Sciences: Wang et al. Proc. Natl. Acad. Sci. USA 95 (1998)

(10 mg͞kg, i.p.). As shown in Fig. 6, CNI-1493 alone (2.5 mg͞kg) fetus or in a tumor), spermine can effectively mediate macro- caused only a 35 Ϯ 12% reduction of serum TNF (P Ͼ 0.05 vs. phage deactivation. It is intriguing to consider that the magnitude vehicle control), whereas fetuin (20 mg͞kg) administered with of the innate immune response reflects a balance between the the same dose of CNI-1493 resulted in a significant reduction of stimulatory activity of CD14 opsonizing LPS and the deactivating TNF (Ͼ74 Ϯ 8%, P ϭ 0.03). activity of fetuin opsonizing spermine. DISCUSSION We thank Dr. Kirk Manogue for critical reading of the manuscript and The overproduction of proinflammatory cytokines by activated Ms. Dee Prieto for excellent secretarial assistance. This research is supported by National Institutes of Health Grant DK 49283 (to K.J.T.) macrophages causes tissue injury in septic shock, rheumatoid and by institutional support from the Picower Institute for Medical arthritis, stroke, allograft rejection, inflammatory bowel disease, Research. trauma, , and other disorders (1, 2). Fetuin has been long recognized as a negative acute-phase protein, but its 1. Wang, H. & Tracey, K. J. (1998) in Inflammation: Basic Principles and Clinical Correlates, eds. Gallin, J., Snyderan, R., Fearon, D., Haynes, B. & Nathan, C. biological role in modulating innate immune responses has not (Lippincott, Philadelphia), Vol. 3, in press. been understood. Fetuin has been implicated as a carrier for 2. Moldawer, L. L. (1994) Crit. Care Med. 22, S3–S7. biologically active compounds (22) and as an inhibitor of insulin 3. Schleimer, R. P. (1993) Eur. J. Clin. Pharmacol. 45, S43–S44. 4. Tsunawaki, S., Sporn, M., Ding, A. & Nathan, C. (1988) Nature (London) 334, receptor tyrosine kinase (34, 35). Fetuin carries a net negative 260–262. charge in the terminal sialic acid residues, as well as in several 5. Bogdan, C. & Nathan, C. (1993) Ann. N.Y. Acad. Sci. 685, 713–739. aspartate͞glutamate-rich domains (such as 11E-P-A͞N-C-D-D- 6. Oswald, I. P., Gazzinelli, R. T., Sher, A. & James, S. L. (1992) J. Immunol. 148, ͞ ͞ ͞ ͞ 28 58 ͞ ͞ ͞ 3578–3582. P V-D E-T-E-A-A-L-A V-V I-D ; E-V L-Y F-D E-I-E- 7. Hess, P. J., Seeger, J. M., Huber, T. S., Welborn, M. B., Martin, T. D., Harward, 75 89 I-D-T-L-E-T-T-C-H-V-L-D ; E-H-A-V-E-G-D-C-D-I͞F- T. R., Duschek, S., Edwards, P. D., Solorzano, C. C., Copeland, E. M. & H͞Q-V͞L-L-K-Q͞L-D104; and 115D-S-S-P-D-S-A-E-D-V-R͞H- Moldawer, L. L. (1997) J. Vasc. Surg. 26, 113–118. K-L͞V-C-P͞Q-D͞N͞R130). It now appears that the sialic acid 8. Randow, F., Syrbe, U., Mazel, C., Krausch, D., Zuckermann, H., Platzer, C. & Volk, H. D. (1995) J. Exp. Med. 181, 1887–1892. moieties of fetuin opsonize spermine and cationic cytokine- 9. Oh-ishi, S., Utsunomiya, I., Yamamoto, T., Komuro, Y. & Hara, Y. (1996) Eur. inhibiting molecules for macrophages. Moreover, the tetravalent J. Pharmacol. 300, 255–259. guanylhydrazone CNI-1493 co-opts this activity of fetuin to gain 10. Szabo, C., Southan, G. J., Thiemermann, C. & Vane, J. R. (1994) Br. J. Pharmacol. 113, 757–766. macrophage cell entry and inhibit cytokine synthesis. Fetuin, a 11. Zhang, M., Caragine, T., Wang, H., Cohen, P. S., Botchkina, G., Soda, K., Bianchi, ubiquitous serum protein, is present on the external surface of M., Ulrich, P., Cerami, A., Sherry, B. & Tracey, K. J. (1997) J. Exp. Med. 185, many cells including neurons, macrophages, and hemopoietic 1759–1768. 12. Koj, A. (1996) Biochim. Biophys. Acta 1317, 84–94. cells (22). In agreement with these observations, we found that 13. Steel, D. M. & Whitehead, A. S. (1994) Immunol. Today 15, 81–88. fetuin is associated predominantly (Ͼ80% of total amount) with 14. Chen, L. M., Chao, L. & Chao, J. (1997) Life Sci. 60, 1431–1435. the macrophage membrane fraction (Western blots not shown). 15. Pedersen, K. O. (1944) Nature (London) 154, 575. 16. Dziegielewska, K. M., Brown, W. M., Casey, S. J., Christie, D. L., Foreman, R. C., Macrophage-associated fetuin is depleted by serum deprivation, Hill, R. M. & Saunders, N. R. (1990) J. Biol. Chem. 265, 4354–4357. which renders macrophages resistant to the deactivating effects of 17. Dziegielewska, K. M., Brown, W. M., Gould, C. C., Matthews, N., Sedgwick, J. E. CNI-1493 and spermine. Considered together, these results now & Saunders, N. R. (1992) J. Comp. Physiol. 162, 168–171. 18. van Oss, C. J., Bronson, P. M. & Border, J. R. (1975) J. Trauma 15, 451–455. suggest that macrophages ‘‘adopt’’ fetuin from serum, a phenom- 19. Lebreton, J. P., Joisel, F., Raoult, J. P., Lannuzel, B., Rogez, J. P. & Humbert, enon previously reported in human embryonic cells (36), G. (1979) J. Clin. Invest. 64, 1118–1129. Sertoli-spermatogenic cells (37), lymphokine-activated killer cells 20. Daveau, M., Christian-Davrinche, Julen, N., Hiron, M., Arnaud, P. & Lebreton, (38), and breast epithelial cells (39). The depletion of fetuin by J. P. (1988) FEBS Lett. 241, 191–194. 21. van Oss, C. J., Gillman, C. F., Bronson, P. M. & Border, J. R. (1974) Immunol. serum-starvation or antibodies in macrophages, however, renders Commun. 3, 329–335. them insensitive to CNI-1493 and spermine. 22. Dizgielewska, K. M. & Brown, W. M., eds. (1995) Fetuin (Landes, Austin, TX). The precise mechanism by which fetuin facilitates entry of 23. Lewis, J. G. & Andre, C. M. (1981) Immunology 42, 481–487. 24. Lewis, J. G. & Andre, C. M. (1981) Immunol. Commun. 10, 541–547. cationic molecules into macrophages remains unclear. No specific 25. Lewis, J. G. & Andre, C. M. (1980) Immunology 39, 317–322. receptor for fetuin has been identified, although receptors for 26. Dziegielewska, K., Brown, W. M., Deal, A., Foster, K. A., Fry, E. J. & Saunders, asialoglycoproteins with exposed terminal galactose or N- N. R. (1996) Histochem. Cell Biol. 106, 319–330. 27. Falquerho, L., Paquereau, L., Vilarem, M. J., Galas, S., Patey, G. & Le Cam, A. acetylglucosamine are known (40–42). Asialoglycoprotein recep- (1992) Nucleic Acids Res. 20, 1983–1990. tors are present on the surface of various cells, including Kupffer 28. Yet, M. G., Chin, C. C. & Wold, F. (1988) J. Biol. Chem. 263, 111–117. cells and macrophages, where they serve to bind, internalize, and 29. Wang, H., Zhang, M., Soda, K., Sama, A. & Tracey, K. J. (1997) Lancet 350, 861–862. dispose of desialylated serum glycoproteins (40–42). It is plau- 30. Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M. R., Zimmerman, G. A., sible that cationic molecules such as spermine or CNI-1493 bind Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., et al. to and neutralize the terminal sialic acid residues of fetuin, and (1995) Mol. Med. 1, 254–266. that the resultant fetuin͞cation complex then can interact with 31. Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Calandra, T., Zhang, X., Bukrinsky, M., et al. (1996) J. Exp. the asialoglycoprotein receptor. Once internalized, CNI-1493 or Med. 183, 927–936. spermine might become available to interact with an unknown 32. Cohen, P. S., Nakshatri, H., Dennis, J., Caragine, T., Bianchi, M., Cerami, A. & intracellular target, leading to inhibition of the translation of Tracey, K. J. (1996) Proc. Natl. Acad. Sci. USA 93, 3967–3971. 33. Cohen, P. S., Schmidtmayerova, H., Dennis, J., Dubrovsky, L., Sherry, B., Wang, proinflammatory cytokine mRNAs (11, 29–31). H., Bukrinsky, M. & Tracey, K. J. (1997) Mol. Med. 3, 339–346. It now appears that the availability of fetuin to macrophages is 34. Brown, W. M., Christie, D. L., Dziegielewska, K. M., Saunders, N. R. & Yang, critical in regulating the innate immune response to tissue injury F. (1992) Cell 68, 7–8. 35. Mathews, S. T., Srinivas, P. R., Leon, M. A. & Grunberger, G. (1997) Life Sci. and infection, because fetuin is required for macrophage deac- 61, 1583–1592. tivation by endogenous cations such as spermine (11, 29). De- 36. Rohrlich, S. T. &. Rifkin, D. B. (1981) J. Cell Physiol. 109, 1–15. creased levels of human fetuin have been observed in patients 37. Abdullah, M., Crowell, J. A., Tres, L. L. & Kierszenbaum, A. L. (1986) J. Cell Physiol. 127, 463–472. with acute lymphocytic leukemia, Hodgkin’s and non-Hodgkin’s 38. Chertov, O. Y., Ermolaeva, M. V., Satpaev, D. K., Saschenko, L. P., Kabanova, lymphomas, rheumatoid arthritis, acute alcoholic hepatitis, O. D., Lukanidin, E. M., Lukjianova, T. I., Redchenko, I. V., Blishchenko, L. Y. chronic active hepatitis, acute and chronic pancreatitis, inflam- & Gnuchev, N. V. (1994) Immunol. Lett. 42, 97–100. matory bowel disease, and trauma (22, 43). Under conditions 39. Gendler, S. J. & Tokes, Z. A. (1984) J. Cell. Biochem. 26, 157–167. 40. Geffen, I. & Spiess, M. (1992) Int. Rev. Cytol. 137, 181–219. where macrophage-associated fetuin levels are decreased, mac- 41. Ozaki, K., Lee, R. T., Lee, Y. C. & Kawasaki, T. (1995) Glycoconjugate J. 12, rophage deactivation by endogenous counter-regulators may be 268–274. impaired, leading to uncontrolled overproduction of proinflam- 42. Kolb-Bachofen, V., Schlepper-Schafer, J., Vogell, W. & Kolb, H. (1982) Cell 29, 859–866. matory cytokines. On the other hand, in cases where macroph- 43. Kalabay, L., Cseh, K., Jakab, L., Prozsonyi, T., Jakab, L., Benedek, S., Fekete, S. age-associated fetuin levels are extremely high (such as in the & Telegdy, L. (1992) Orv. Hetil. 133, 1553–1560. 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