Soluble CD93 Induces Differentiation of and Enhances TLR Responses Jae-Won Jeon, Joon-Goo Jung, Eui-Cheol Shin, Hye In Choi, Ho Youn Kim, Mi-La Cho, Sun-Wha Kim, This information is current as Young-Soon Jang, Myung-Ho Sohn, Ji-Hyun Moon, of September 27, 2021. Young-Hun Cho, Kwang-Lae Hoe, Yeon-Soo Seo and Young Woo Park J Immunol 2010; 185:4921-4927; Prepublished online 22

September 2010; Downloaded from doi: 10.4049/jimmunol.0904011 http://www.jimmunol.org/content/185/8/4921

Supplementary http://www.jimmunol.org/content/suppl/2010/09/20/jimmunol.090401 http://www.jimmunol.org/ Material 1.DC1 References This article cites 28 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/185/8/4921.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Soluble CD93 Induces Differentiation of Monocytes and Enhances TLR Responses

Jae-Won Jeon,*,† Joon-Goo Jung,† Eui-Cheol Shin,‡ Hye In Choi,† Ho Youn Kim,x Mi-La Cho,x Sun-Wha Kim,† Young-Soon Jang,† Myung-Ho Sohn,† Ji-Hyun Moon,† Young-Hun Cho,‡ Kwang-Lae Hoe,† Yeon-Soo Seo,* and Young Woo Park†

The cell surface CD93 is known to be involved in the regulation of and . Although typically membrane-bound, a soluble form of CD93 (sCD93) has recently been identified. Currently, however, the role of sCD93 in mono- cyte function is unknown. In the current study, we analyzed the functional effects of sCD93 on THP-1 monocytic cells and human primary monocytes. Various forms of recombinant human sCD93 were used to investigate the effects of this molecule on both human primary monocytes and a monocytic cell line, THP-1. We found that sCD93 induced differentiation of monocytes to -like cells, as evidenced by activated cell adhesion and increased phagocytic activities. In addition, this differ- Downloaded from entiation resulted in an enhanced response to TLR stimulation in terms of differentiation marker expression and proinflam- matory cytokine production. Furthermore, sCD93 enhanced LPS-stimulated TNF-a production even prior to differentiation. To investigate a possible role for sCD93 in the pathogenesis of chronic inflammatory diseases, we assessed the concentration of sCD93 in synovial fluid from patients with rheumatoid arthritis and found it to be significantly increased compared with synovial fluid from patients with osteoarthritis. Together, these data revealed a function for sCD93 that may have implications in inflammation and inflammatory diseases including rheumatoid arthritis. The Journal of Immunology, http://www.jimmunol.org/ 2010, 185: 4921–4927.

he type 1 transmembrane glycoprotein CD93 (C1qRP) is CD93 was originally shown to play a role in the regulation of encoded by the CD93 found on 20, C1q-mediated phagocytosis in an inhibition assay through use of T position p11.21 in humans (1–3). CD93 consists of a signal anti-CD93 mAbs (8). In further studies, these mAbs were also peptide, a C-type carbohydrate-recognition domain (CRD), five shown to inhibit phagocytosis mediated by mannose-binding epidermal growth factor (EGF)-like domains, a mucin domain, a and pulmonary surfactant protein, indicating a critical role for single transmembrane domain, and an intracellular domain with a CD93 in the regulation of phagocytosis. Indeed, CD93-null mice by guest on September 27, 2021 potential tyrosine kinase phosphorylation site (2, 4–8). CD93 ho- displayed impaired clearance of apoptotic cells, which is medi- mologs in mouse and rat are known as AA4 and are 67–87% iden- ated by phagocytic myeloid cells. Furthermore, immobilized anti- tical (rat versus mouse: 87%; human versus mouse: 67%; human CD93 IgM mAb triggered phagocytosis in vitro (5, 17). However, versus rat: 77%) to the human isoform (9–12). Expression of the mechanism by which CD93 regulates phagocytosis remains CD93 occurs in various cell types including myeloid cells, hema- unclear. topoietic stem cells, NK cells, , , and endothe- In addition to its role in phagocytosis, CD93 is known to be lial cells, but not in tissue (2, 12–16). involved in the regulation of cell adhesion. Ligation of CD93 with an anti-CD93 mAb induced homotypic aggregation of LPS- stimulated U937 cells and triggered rapid spreading of HUVEC *Nucleic Acid Biochemistry Laboratory, Department of Biological Science and ‡Laboratory of Immunology and Infectious Diseases, Graduate School of Medical cells in vitro (18, 19). This suggests that the expression of CD93 Science and Engineering, Korea Advanced Institute of Science and Technology; on endothelial cells and myeloid cells might play a role in the †Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon; and xThe Rheumatism Research Center, Catholic Research regulation of cell adhesion and in the homing of inflammatory Institute of Medical Science, The Catholic University of Korea, Seoul, Republic of cells (6, 15, 20). Korea Recently, two separate studies reported the identification of Received for publication December 15, 2009. Accepted for publication August 13, a soluble form of CD93 (sCD93) (7, 21). Initially, it was dem- 2010. onstrated that hypoglycosylation of CD93 caused an increase in This work was supported by grants from the Ministry of Education, Science and the levels of sCD93 in culture medium. This suggested that sCD93 Technology (NBM4700912) and the Ministry of Knowledge Economy (Regional R&D Cluster Project; B0009735) of Korea. was proteolytically cleaved from the surface-bound form (7). Fur- Address correspondence and reprint requests to Young Woo Park or Yeon-Soo Seo, ther studies showed that CD93 was rapidly shed from the surface Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Re- of human myeloid cells, and sCD93 was detected in human plasma public of Korea (Y.W.P.) or Nucleic Acid Biochemistry Laboratory, Department of (21, 22). Additionally, Greenlee et al. (23) demonstrated produc- Biological Science, KAIST, Daejeon 305-701, Republic of Korea (Y.-S.S.). E-mail addresses: [email protected] (Y.W.P.) or [email protected] (Y.-S.S.) tion of sCD93 in vivo under inflammatory conditions. However, the The online version of this article contains supplemental material. function of sCD93 remains to be defined. Abbreviations used in this paper: CRD, carbohydrate-recognition domain; EGF, epi- In the current study, we synthesized various forms of recom- dermal growth factor; K/O, knockout; OA, osteoarthritis; RA, rheumatoid arthritis; binant human sCD93 to elucidate the functional effects of sCD93. RFP, red fluorescent protein; sCD93, soluble form of CD93; SEAP, secreted embry- Specifically, we analyzed the effects of sCD93 on differentiation onic alkaline phosphatase. of monocytes and on TLR responses by using human primary Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 monocytes and a monocytic cell line. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904011 4922 THE FUNCTION OF SOLUBLE CD93

Materials and Methods in methanol, stained with 10% (v/v) solution of Giemsa (Sigma-Aldrich), Reagents and cell culture and the number of adherent cells was evaluated over 10 fields. For each well, the ratio between the number of cells in 10 fields after plating (NT) LPS, THP-1 Blue cells (human monocytic leukemia line with an NF-kB– and the number of adherent cells in 10 fields after culturing and washing 3 inducible reporter), TLR agonists, and QUANTI-Blue detection medium (NA) was defined as the percent adherence (NA/NT 100). To measure were purchased from Invivogen (San Diego, CA). PMA was purchased phagocytosis, human primary monocytes were differentiated for 24 h in the from Sigma-Aldrich (St. Louis, MO). Normal human IgG control protein presence of hsCD93-Fc, hsCD93-His, or controls. Then, cells were in- was purchased from Jackson ImmunoResearch Laboratories (West Grove, cubated for 6 h with Escherichia coli that expressed monomeric red PA). Mouse anti-human CD93 mAb and goat anti-human CD93 Ab were fluorescent protein (RFP). The cells were washed with cell culture medium purchased from R&D Systems (Minneapolis, MN). FITC-conjugated Abs without FBS, and the fluorescence intensity was assessed by flow cy- against CD1a, CD11b, and control IgG were purchased from BD Bio- tometry. The cells were also examined on a confocal laser scanning mi- sciences (San Jose, CA). FITC-conjugated anti-His Ab was purchased from croscope (LSM 510; Carl Zeiss, Oberkochen, Germany). A Z-stack of 40 Invitrogen (Carlsbad, CA). Human monocytic leukemia THP-1 cells were images was acquired with a 2-mm interval. Images were analyzed using the purchased from American Type Culture Collection (Manassas, VA). The Zeiss LSM software package (Carl Zeiss, Oberkochen, Germany). THP-1 cells and human primary monocytes were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin G (100 Assessment of cytokine production IU/ml), streptomycin (100 mg/ml), L-glutamine (2 mM), HEPES (10 mM), and sodium pyruvate (1.0 mM). THP-1 cells were incubated with hsCD93-Fc or controls for 48 h and further incubated with or without LPS for 24 h. The culture supernatants were Preparation of human primary monocytes collected and cytokine levels were examined with the Quantikine assay (R&D Systems) according to the manufacturer’s instructions. Cytokine PBMCs were isolated from venous blood of normal donors by Ficoll density production was also assessed in primary human monocyte cultures. Human gradient, and primary monocytes were subsequently isolated with CD14 mag- primary monocytes were rested for 24 h, incubated with hsCD93-Fc, Downloaded from netic beads (Miltenyi Biotec, Auburn, CA). The purity of the isolated human hsCD93-His, or controls for 48 h, and further incubated with or without primary monocytes was verified with anti–CD14-FITC Ab (BD Biosciences) LPS for 24 h. The culture supernatants were collected, and cytokine levels by flow cytometry (Beckman Coulter, Fullerton, CA), and it was 90–94%. were examined with the Quantikine assay kit (R&D Systems). Production of recombinant humansCD93-Fc Secreted embryonic alkaline phosphatase reporter assay Four different forms of recombinant human sCD93 were constructed into pcDNA3.1 vector as follows (Supplemental Fig. 1): 1) human IgG1 Fc THP-1 blue cells express the whole set of TLRs, from TLR1–TLR10, and (named Fc only); 2) CRD and EGF domains of human CD93 (aa 24–470) they contain a reporter plasmid that expresses a secreted embryonic al- http://www.jimmunol.org/ fused with human IgG1 Fc (named hsCD93-Fc); 3) CRD, EGF, and mucin kaline phosphatase (SEAP) under the control of NF-kB and AP-1 tran- domains of human CD93 (aa 24–552) fused with human IgG1 Fc (named scription factors. THP-1 Blue cells were incubated with hsCD93-Fc or hsCD93-Mucin-Fc); and 4) CRD and EGF domains of human CD93 (aa controls for 48 h and further incubated with or without a TLR agonist 24–470) fused with 6x His (named hsCD93-His). Briefly, for the Fc only for 24 h. The following TLR agonists were used: Pam3CSK4 (0.1 mg/ml) 8 construction, a leader sequence (gene ID: K02149; protein: AAA51633; for TLR1/2, HKLM (10 cell) for TLR2, LPS E. coli K12 (1.0 mg/ml) for mouse IgG H chain, primer set: 59-ATAGGCTAGCGCCACCATGGGAT- TLR4, flagellin (1.0 mg/ml) for TLR5, FSL-1 (0.2 mg/ml) for TLR6/2, and GG-39,59-TGTGTGAGTTTTGTCCGAGTGGACATCTGT-39) and a human ssRNA40 (5.0 mg/ml) for TLR8. To quantify secreted SEAP, the culture IgG1 Fc region (primer set: 59-ACAGATGTCCACTCGGACAAAACTCA- supernatant was incubated with the QUANTI-Blue colorimetric assay re- CACA-39,59-CCAGCTCGAGCTATTTACCCGGAGACAG-39) were am- agent (Invivogen) for 24 h at 37˚C. The OD at 655 nm was evaluated with plified, and the overlap extension PCR was performed. For the hsCD93-Fc a VERSAmax tunable microplate reader (Molecular Devices, Toronto, On- by guest on September 27, 2021 construction, the leader sequence (primer set: 59-ATAGGCTAGCGCCAC- tario, Canada). All assays were run in triplicate. CATGGGATGG-39,59-CGCCTCCGTGTCAGCCGAGTGGACATCTGT-39), aa 24–470 of human CD93 (primer set: 59-ACAGATGTCCACTCGGCT- Real-time quantitative PCR GACACGGAGGCG-39,59-TGTGTGAGTTTTGTCCCCCATGGTGCAAGA- The CFX96 system (Bio-Rad, Hercules, CA) was used for real-time PCR 39), and the human IgG1 Fc region (primer set: 59-TCTTGCACCATGGGG- analysis. Thermal cycling was performed as follows: initial denaturation at GACAAAACTCACACA-39,59-CCAGCTCGAGCTATTTACCCGGAGA- 95˚C for 3 min followed by 40–45 cycles of 95˚C for 15 s and 60˚C for 1 CAG-39) were amplified, and the overlap extension PCR was performed. For min. For each sample, 2 ml cDNA was added to 23 ml iQ SYBR Supermix the hsCD93-Mucin-Fc construction, the leader sequence (primer set: 59- (Bio-Rad) containing a primer pair, and each reaction was performed in ATAGGCTAGCGCCACCATGGGATGG-39,59-CGCCTCCGTGTCAGCC- duplicate. The standard comparative cycle threshold method was used to GAGTGGACATCTGT-39), aa 24–552 of CD93 (primer set: 59-ACAG- determine the relative gene expression levels, and the cycle threshold value ATGTCCACTCGGCTGACACGGAGGCG-39,59-TGTGTGAGTTTTG- of each sample was normalized to GAPDH. The corresponding primer TCGGCGTGATGGATGCT-39), and the human IgG1 Fc region (primer set: pairs of TLR2, TLR4, MYD88, and GAPDH were purchased from Bioneer 59-AGCATCCATCACGCCGACAAAACTCACACA-39,59-CCAGCTCGAG- (Daejeon, Republic of Korea). CTATTTACCCGGAGACAG-39) were amplified, and the overlap exten- sion PCR was performed. All PCR products were digested with NheI and XhoI and inserted into pcDNA3.1 vector. For hsCD93-His construction, Sandwich ELISA for sCD93 PCR amplification (the leader sequence and aa 24–470 of CD93) was A 96-well micro assay plate (BD Biosciences) was coated with mouse anti- 9 performed with a primer set (5 -ATAGGCTAGCGCCACCATGGGATGG- human CD93 MAb (2 mg/ml) and then blocked with 3% skim milk. Human 9 9 3 ,5-CTGGCTCGAGCTAATGATGATGATGATGATGCCCCATGGTG- synovial fluid was added and incubated for 2 h. After washing, the plate 9 CAAGA-3 ) using the template of the construct hsCD93-Fc. The PCR was incubated with goat anti-human CD93 Ab (1 mg/ml) for 1 h. Sub- product was digested with NheI and XhoI and inserted into pcDNA3.1 sequently, the plate was incubated with anti-goat IgG-HRP for 1 h. After vector. HsCD93-Fc and hsCD93-Mucin-Fc were expressed in HEK293E washing, the plate was colorimetrically developed with o-phenylenedi- cells and purified on a protein A-Sepharose column (Amersham Bio- amine dihydrochloride substrate (Sigma-Aldrich), and absorbances were sciences, Sunnyvale, CA) according to the manufacturer’s instructions. read with the VERSAmax tunable microplate reader (Molecular Devices). Human IgG1 Fc without the CD93 domain was also expressed and purified with same methods and used as a negative control. HsCD93-His was ex- pressed in HEK293E cells and purified on a Ni-NTA Agarose column Results (Qiagen, Valencia, CA) according to the manufacturer’s instructions. All sCD93 induces differentiation of monocytes to macrophage- of the purified recombinant were dialyzed with PBS, analyzed by SDS-PAGE, and analyzed with the Limulus amebocyte lysate test kit (Cape like cells Cod, East Falmouth, MA) to examine the endotoxin level; the concen- To study the functional role of sCD93 on monocytes, we prepared , tration of endotoxin was 1–3 EU/ml. recombinant forms of human sCD93. As the domain structure of Cell adhesion assay and phagocytosis assay naturally shed sCD93 is not clear, we constructed four versions of THP-1 cells (1 3 104 cells/well) were plated in a 96-well culture plate and recombinant sCD93: Fc only, hsCD93-Fc, hsCD93-Mucin-Fc, and treated with hsCD93-Fc for 1 h. Next, the plate was washed three times hsCD93-His (Fig. 1A,1B). Using a functional assay, we evaluated with PBS, and microscopic photographs were taken. The cells were fixed the role of the mucin domain by comparing hsCD93-Fc and The Journal of Immunology 4923 Downloaded from

FIGURE 1. Preparation of recombinant human sCD93. A, Schematic diagram of recombinant human sCD93 proteins. B, SDS-PAGE analysis of purified recombinant human sCD93. Four types of recombinant human sCD93 were expressed in HEK 293 cells and then purified. Denatured protein samples were http://www.jimmunol.org/ separated on 4–20% gradient SDS-PAGE. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue. C, THP-1 cells were fixed with 4% paraformaldehyde for 10 min, blocked with 5% normal goat serum for 30 min, incubated with hIgG control or hsCD93-Fc, and further incubated with FITC-conjugated anti-human IgG. Flow cytometry results show the specific binding of hsCD93-Fc to THP-1 cells. D, Using the same method described in C, human primary monocytes were fixed with 4% paraformaldehyde for 10 min, blocked with 5% normal goat serum for 30 min, incubated with Fc control, hsCD93-Fc, or hsCD93-Fc mixed with goat anti-hCD93 polyclonal Ab, and further incubated with FITC-conjugated anti-human IgG. The flow cytometry results show the specific binding of hsCD93-Fc to human primary monocytes. E, Human primary monocytes were fixed with 4% paraformaldehyde for 10 min, blocked with 5% normal goat serum for 30 min, incubated with hsCD93-His, and further incubated with FITC-labeled anti-His Ab. Flow cytometry results show the specific binding of hsCD93-His to cells. by guest on September 27, 2021 hsCD93-Mucin-Fc (Supplemental Fig. 2) and found similar ef- cytes with monomeric RFP-expressing E. coli. Subsequently, fluo- fects on cytokine production, indicating that the mucin domain is rescent microscopy and flow cytometry were performed to detect dispensable for function of sCD93. In the current study, therefore, the RFP signals from phagocytosed E. coli (Fig. 2C, Supplemental we used hsCD93-Fc and hsCD93-His for all functional assays. Fig. 5). To differentiate true phagocytosis from bacterial adher- We began our studies by determining whether sCD93 could bind ence to the cell surface, we performed a Z-stack analysis of to human monocytes, as the receptor expression patterns for CD93 confocal microscopy images and confirmed that ingested bacteria are unclear. We found that hsCD93-Fc was able to bind to the cell were located in the cytoplasm of monocytes (Supplemental Fig. surface of THP-1 cells (Fig. 1C) and human primary monocytes 6). Human primary monocytes treated with hsCD93-Fc for 3 d (Fig. 1D), thus indicating that monocytes express a putative re- exhibited enhanced phagocytic activity (Fig. 2C). Moreover, anti- ceptor for sCD93. Moreover, this binding was partially blocked by CD93 Ab partially abrogated the effect of hsCD93-Fc on the the addition of anti-CD93 Ab (Fig. 1D). In addition, hsCD93-His phagocytic activity, illustrating the specificity of the response. In was also able to bind the cell surface of human primary mono- addition, heat-denatured hsCD93-Fc did not enhance the phago- cytes, indicating that the His tag did not interfere with hsCD93– cytic activity of monocytes. Although the effect was less than that receptor interactions (Fig. 1E). observed for the Fc form, hsCD93-His was also shown to enhance Next, we tested the effect of hsCD93-Fc on cell adherence, phagocytic activity (Fig. 2D). Taken together, these data suggest a functional indicator of macrophage differentiation (24). We that sCD93 induces the differentiation of monocytes to macro- found that hsCD93-Fc could induce THP-1 cell adhesion within phage-like cells, as evidenced by enhanced cell adhesion and in- 1 h in a dose-dependent manner (Fig. 2A, Supplemental Fig. 3). In creased phagocytic activities. addition, monomeric sCD93 without the Fc region (hsCD93-His) was also able to induce cell adherence, although the effect was less potent than that of hsCD93-Fc. After 3 d of culture in the pre- Differentiation of cells by sCD93 increases their sensitivity to sence of hsCD93-Fc, THP-1 cells exhibited a flattened morphol- TLR stimulation ogy with extensive pseudopodia (Fig. 2B). This macrophage-like We studied the biological function of sCD93 by assessing its effects morphological change was also observed with PMA treatment. A on monocyte differentiation and cytokine production. For this similar morphology was observed in freshly isolated human mono- purpose, THP-1 cells were cultured with or without hsCD93-Fc for cytes after a shorter period (24 h) (Supplemental Fig. 4). 48 h and subsequently stimulated with LPS for an additional 24 h. As morphological changes are indicative of monocyte differ- These time points were determined in a set of preliminary ex- entiation, we next determined whether hsCD93-Fc could enhance periments in which we evaluated the effects of sCD93 at various phagocytic activity, a functional property of mature macrophages. times and found the most pronounced cellular responses at 48 h Phagocytosis was evaluated by coculturing human primary mono- (Supplemental Fig. 7). 4924 THE FUNCTION OF SOLUBLE CD93 Downloaded from http://www.jimmunol.org/

FIGURE 2. Human sCD93 enhanced cell adhesion and phagocytosis in THP-1 cells and human primary monocytes. A, Adherent THP-1 cells were counted posttreatment of hsCD93-Fc (5, 1, and 0.2 mg/ml), hsCD93-His (5, 1, and 0.2 mg/ml), or controls (5 mg/ml) and expressed as a percentage of the total number of cells cultured. All experiments were conducted in triplicate. Data represent the mean 6 SEM. Similar results were obtained from three independent experiments. B, Phase-contrast images of THP-1 cells treated with Fc control (5 mg/ml), hIgG control (5 mg/ml), hsCD93-Fc (5 mg/ml), or PMA (0.1 mg/ml) for 3 d (original magnification 3200). C, Human primary monocytes were cultured for 24 h in the presence of Fc control, hsCD93-Fc, heat-denatured (at 95˚C for 30 min) hsCD93-Fc, or hsCD93-Fc mixed with goat anti-CD93 polyclonal Ab and then incubated for 6 h with E. coli labeled with RFP. Flow cytometric data indicate the extent of phagocytosis. The data are representative of two independent experiments. D, Human primary monocytes were cultured for 24 h in the presence of Fc control, hsCD93-Fc, or hsCD93-His and then incubated for 6 h with E. coli labeled with RFP. Flow by guest on September 27, 2021 cytometric data indicate the extent of phagocytosis. The data are representative of three independent experiments. pp , 0.05; ppp , 0.01.

Expression of the differentiation markers CD1a and CD11b Therefore, this suggests that another mechanism is responsible for was marginally increased by hsCD93-Fc alone and moderately the enhanced sensitivity to TLR stimulation. increased by LPS alone (Fig. 3A). Intriguingly, pretreatment with Effect of sCD93 on undifferentiated monocytes hsCD93-Fc greatly enhanced the LPS-induced increases in CD1a and CD11b expression (18.2-fold and 5.06-fold, respectively). A By a series of experiments, we showed that sCD93 induces dif- similar phenomenon was observed for cytokine production. When ferentiation of monocytes and that the differentiation results in evaluating the proinflammatory cytokines TNF-a, IL-1b, and IL-6 increased sensitivity to TLR stimulation. Next, we studied if sCD93 (Fig. 3B), we found that pretreatment with hsCD93-Fc enhanced enhances TLR responses of monocytes even without induction of their LPS-stimulated production. Similar results were observed differentiation. To clarify this issue, we examined LPS-stimulated when monomeric recombinant sCD93 (hsCD93-His) was used TNF-a production from THP-1 monocytes with or without sCD93 (Fig. 3C). In particular, hsCD93-His alone slightly induced TNF-a cotreatment (Fig. 5). Both hsCD93-Fc and hsCD93-His signifi- production. In addition, anti-CD93 Ab completely abrogated cantly enhanced LPS-stimulated TNF-a production when coadmin- hsCD93-Fc–induced TNF-a production (Fig. 3D). These results istered with LPS. Thus, soluble CD93 synergizes with LPS, increas- were confirmed in human primary monocytes (Fig. 3E). Taken ing TNF-a production even without monocyte differentiation. together, these results indicate that hsCD93-Fc increased the sen- sitivity of monocytes to LPS in terms of both differentiation and sCD93 was increased in synovial fluid of patients with cytokine production. rheumatoid arthritis Next, we explored the effect of hsCD93-Fc on the response to To elucidate possible clinical implications of sCD93 production, TLR agonists other than LPS through use of a reporter system. we studied synovial fluid from patients with rheumatoid arthritis THP-1 Blue cells secrete the protein SEAP into the culture me- (RA) and compared it to that from patients with osteoarthritis (OA), dium upon TLR agonist-induced NF-kB activation. We found that a noninflammatory disease control. sCD93 was more abundant in hsCD93-Fc increased the sensitivity of THP-1 Blue cells to sev- the synovial fluid of patients with RA than in synovial fluid from eral TLR agonists including those specific for TLR2, TLR4, patients with OA (Fig. 6A). However, in plasma, there was no sig- TLR5, TLR6, and TLR8, but not the agonist for TLR1 (Fig. 4). To nificant difference observed, although the mean value was higher probe the mechanism underlying this effect, we investigated in the plasma of patients with RA (Fig. 6B). These data indicate whether hsCD93-Fc might affect the expression of the TLRs or that CD93 might play a role in the pathogenesis of inflammatory downstream molecule MyD88 by conducting a real-time PCR diseases. Moreover, sCD93 might be considered as a biomarker study. However, we found no evidence of either (data not shown). for chronic inflammatory diseases, such as RA. The Journal of Immunology 4925 Downloaded from http://www.jimmunol.org/

FIGURE 3. hsCD93-Fc pretreatment increased the sensitivity of monocytes to LPS. A, THP-1 cells were cultured with hsCD93-Fc (5 mg/ml) or hIgG control (5 mg/ml) for 48 h. Subsequently, cells were incubated with or without 1 mg/ml of LPS for an additional 24 h. Cells were collected and labeled with anti–CD1a-FITC or anti–CD11b-FITC. Flow cytometry data indicate the expression of the differentiation markers CD1a and CD11b. Mean fluorescence intensity was indicated in each histogram. B, THP-1 cells were cultured with hsCD93-Fc (5 mg/ml) or controls (5 mg/ml) for 48 h. Subsequently, cells were incubated with or without 1 mg/ml of LPS for an additional 24 h. Culture supernatants from THP-1 cells were collected and the concentration of TNF-a, IL- 1b, and IL-6 were determined with a Quantikine assay kit. Data represent the mean 6 SEM. Similar results were obtained from three independent experiments. C, In the same setting, THP-1 cells were treated with Fc only (5 mg/ml), hsCD93-Fc (5 mg/ml), or hsCD93-His (1 mg/ml), and the con- by guest on September 27, 2021 centration of TNF-a was determined. Data represent the mean 6 SEM. Similar results were obtained from three independent experiments. D, In the same setting, THP-1 cells were treated with Fc only (1 mg/ml), hsCD93-Fc (1 mg/ml), or hsCD93-Fc (1 mg/ml) in combination with goat anti-hCD93 polyclonal Ab (2 mg/ml), and the concentration of TNF-a was determined with a Quantikine assay kit. Data represent the mean 6 SEM. Similar results were obtained from two independent experiments. E, Primary human CD14+ monocytes were isolated and cultured with hsCD93-Fc or Fc control at indicated various concentrations for 48 h. Subsequently, cells were incubated with or without 1 mg/ml LPS for an additional 24 h. Culture supernatants were collected, and the concentrations of TNF-a, IL-1b, and IL-6 were determined. Data represent the mean 6 SEM. Similar results were obtained from three independent experiments. pp , 0.05; ppp , 0.01.

Discussion Our data also support a role for sCD93 as a promoter of in- The existence of a sCD93 was only recently discovered, and its flammatory responses, because we observed increased cytokine biological functions have not yet been elucidated. In this study, we production in response to LPS stimulation in monocytes that had analyzed various forms of recombinant human sCD93 to evaluate the been pretreated with sCD93 (Figs. 3, 4). However, Harhausen effects of this molecule on monocyte differentiation and function et al.’s (28) recent study using the cerebral ischemia model (Fig. 1). We determined that sCD93 induced both monocyte adher- showed increased leukocyte infiltration and CCL21 expression in ence and morphological changes and enhanced phagocytosis (Fig. 2), CD93 knockout (K/O) mice compared with wild-type mice, sug- suggesting that sCD93 is able to induce monocyte differentiation. gesting that CD93 acts to downmodulate inflammatory responses. Increased monocyte phagocytosis following incubation with The discrepancy between their study and our current study may be sCD93 is consistent with the previous observation that CD93-null explained by the difference in pathological conditions. Specifi- mice exhibit a defect in the clearance of apoptotic cells (5, 25), as cally, cerebral ischemia induces inflammatory responses by caus- the scavenging of apoptotic cells is known to be achieved through ing tissue damage, and this may produce qualitatively different phagocytosis (26, 27). Thus, defective clearance of apoptotic cells results than inflammatory responses generated via introduction of in CD93-null mice might actually be caused by the lack of sCD93. microbiological substances such as LPS. Thus, it is possible that Very recently, Greenlee et al. (23) reported the induction of sCD93 CD93 might play different roles in microbiological inflammation in mouse peritoneal lavage fluid after thioglycollate injection. In versus sterile inflammation. Another possible explanation is that addition, they demonstrated that peritoneal macrophage phagocy- in a K/O model system, the absence of CD93 during development tosis was enhanced by the thioglycollate-treated peritoneal lavage may change the physiology of the K/O animal by inducing fluid from CD93 wild-type mice but not that from CD93-null overexpression of compensatory molecules such as CCL21. To mice. This report corroborates our finding that sCD93 enhances clarify this issue, the use of conditional CD93 K/O mice would be monocytic cell phagocytosis. warranted. 4926 THE FUNCTION OF SOLUBLE CD93 Downloaded from

FIGURE 5. Human sCD93 cotreatment with LPS increased the secretion http://www.jimmunol.org/ of TNF-a. THP-1 cells were cultured with Fc only (5 mg/ml), hsCD93-Fc (5 mg/ml) with or without LPS (1 mg/ml), hsCD93-His (1 mg/ml) with or without LPS (1 mg/ml), or LPS only (1.0 mg/ml) for 48 h. Culture super- natants from THP-1 cells were collected at the indicated times and the con- centration of TNF-a was determined. The one result was presented in two separate columns, A (Fc form) and B (His form), to easily distinguish each line. Data represent the mean 6 SEM. Similar results were obtained from two independent experiments. by guest on September 27, 2021 (J.-W. Jeon, unpublished observations). However, it remains to be clarified whether ERK and p38 pathways are the major signaling pathways involved in mediating the effects of hsCD93-Fc. FIGURE 4. hsCD93-Fc pretreatment increased the sensitivity of mono- cytes to other TLR agonists. Poststimulation of THP-1 Blue cells with hsCD93-Fc (5 mg/ml) or controls (5 mg/ml) for 48 h, the cells were treated with the indicated TLR agonists for an additional day. Culture super- natants were assessed for SEAP reporter activity with the QUANTI-blue reagent (Invivogen) to evaluate the degree of NF-kB activation by TLR agonists.

As mentioned above, we also determined that sCD93 increased monocyte sensitivity to LPS and other TLR agonists, as demon- strated by increases in differentiation marker expression, cytokine production, and NF-kB reporter activity (Figs. 3–5). To explore a potential mechanism for these observations, we investigated the expression of TLRs and MyD88 after hsCD93-Fc treatment and found them to remain stable (data not shown). Hence, the mech- anism by which sCD93 exerts this function remains unknown. Future studies will be directed at identification of the cellular receptor for sCD93 and the downstream signaling pathways. In initial attempts to identify the cellular receptor for sCD93, we have isolated sCD93-binding proteins by immunoprecipitation and identified the binding proteins using MALDI-TOF. This process yielded the sequences of several putative receptor proteins (Y.-S. Jang and J.-W. Jeon, unpublished observations), and we are cur- rently attempting to identify which protein serves as a receptor for FIGURE 6. sCD93 was overexpressed in RA synovial fluid. The con- sCD93. We have also examined cell signaling pathways affected centrations of sCD93 in synovial fluid (A) and plasma (B) from patients by hsCD93-Fc treatment. Interestingly, the phosphorylation of with RA and OA were determined by sandwich ELISA. Statistical analysis ERK and p38 was specifically induced by hsCD93-Fc treatment was performed using a two-tailed paired Student t test. pp , 0.01. The Journal of Immunology 4927

In the current study, we demonstrated that synovial fluid from 10. Kim, T. S., M. Park, R. R. Nepomuceno, G. Palmarini, S. Winokur, C. A. Cotman, patients with RA contained higher levels of sCD93 than that from U. Bengtsson, and A. J. Tenner. 2000. Characterization of the murine homolog of C1qR(P): identical cellular expression pattern, chromosomal location and patients with OA (Fig. 6). Although it is unknown whether the functional activity of the human and murine C1qR(P). Mol. Immunol. 37: 377– overexpression of CD93 or sCD93 plays a role in the development 389. of RA, these results suggest a potential clinical application for 11. Dean, Y. D., E. P. McGreal, H. Akatsu, and P. Gasque. 2000. Molecular and cellular properties of the rat AA4 antigen, a C-type lectin-like receptor with CD93. First, sCD93 could be a potential biomarker for RA. How- structural homology to . J. Biol. Chem. 275: 34382–34392. ever, further study will be required to determine if there is a cor- 12. Løvik, G., J. T. Vaage, E. Dissen, C. Szpirer, J. C. Ryan, and B. Rolstad. 2000. relation between the concentrations of sCD93 in the synovial fluid Characterization and molecular cloning of rat C1qRp, a receptor on NK cells. Eur. J. Immunol. 30: 3355–3362. and the severity of RA. Second, sCD93 could be a potential target 13. Nepomuceno, R. R., and A. J. Tenner. 1998. C1qRP, the C1q receptor that for developing new RA treatments. Future studies to determine enhances phagocytosis, is detected specifically in human cells of myeloid line- age, endothelial cells, and platelets. J. Immunol. 160: 1929–1935. whether sCD93 is involved in the pathogenesis of RA are certainly 14. Danet, G. H., J. L. Luongo, G. Butler, M. M. Lu, A. J. Tenner, M. C. Simon, and warranted. D. A. Bonnet. 2002. C1qRp defines a new human stem cell population with In summary, we identified a functional role for sCD93 and hematopoietic and hepatic potential. Proc. Natl. Acad. Sci. USA 99: 10441– 10445. revealed the presence of sCD93 in the chronic inflammatory dis- 15. Fonseca, M. I., P. M. Carpenter, M. Park, G. Palmarini, E. L. Nelson, and eases RA. In the future, detailed studies will be warranted to spe- A. J. Tenner. 2001. C1qR(P), a myeloid cell receptor in blood, is predominantly cifically investigate the mechanism of action of sCD93 and the expressed on endothelial cells in human tissue. J. Leukoc. Biol. 70: 793–800. 16. Webster, S. D., M. Park, M. I. Fonseca, and A. J. Tenner. 2000. Structural and role of sCD93 in the pathophysiology of chronic inflammatory functional evidence for microglial expression of C1qR(P), the C1q receptor that diseases. enhances phagocytosis. J. Leukoc. Biol. 67: 109–116.

17. Guan, E., S. L. Robinson, E. B. Goodman, and A. J. Tenner. 1994. Cell-surface Downloaded from protein identified on phagocytic cells modulates the C1q-mediated enhancement Disclosures of phagocytosis. J. Immunol. 152: 4005–4016. The authors have no financial conflicts of interest. 18. Ikewaki, N., and H. Inoko. 1996. Development and characterization of a novel monoclonal antibody (mNI-11) that induces cell adhesion of the LPS-stimulated human monocyte-like cell line U937. J. Leukoc. Biol. 59: 697–708. 19. Ikewaki, N., H. Tamauchi, A. Yamada, N. Mori, H. Yamao, H. Inoue, and References H. Inoko. 2000. A unique monoclonal antibody mNI-11 rapidly enhances spread 1. Malhotra, R., A. C. Willis, J. C. Jensenius, J. Jackson, and R. B. Sim. 1993. J. Clin. Immunol.

formation in human umbilical vein endothelial cells. 20: 317– http://www.jimmunol.org/ Structure and homology of human C1q receptor (collectin receptor). Immunol- 324. ogy 78: 341–348. 20. Dean, Y. D., E. P. McGreal, and P. Gasque. 2001. Endothelial cells, mega- 2. Nepomuceno, R. R., A. H. Henschen-Edman, W. H. Burgess, and A. J. Tenner. karyoblasts, platelets and alveolar epithelial cells express abundant levels of the 1997. cDNA cloning and primary structure analysis of C1qR(P), the human C1q/ mouse AA4 antigen, a C-type lectin-like receptor involved in homing activities MBL/SPA receptor that mediates enhanced phagocytosis in vitro. Immunity 6: and innate immune host defense. Eur. J. Immunol. 31: 1370–1381. 119–129. 21. Bohlson, S. S., R. Silva, M. I. Fonseca, and A. J. Tenner. 2005. CD93 is rapidly 3. Steinberger, P., A. Szekeres, S. Wille, J. Stockl, N. Selenko, E. Prager, G. Staffler, ¨ shed from the surface of human myeloid cells and the soluble form is detected in O. Madic, H. Stockinger, and W. Knapp. 2002. Identification of human CD93 human plasma. J. Immunol. 175: 1239–1247. as the phagocytic C1q receptor (C1qRp) by expression cloning. J. Leukoc. Biol. 22. Ikewaki, N., H. Tamauchi, and H. Inoko. 2007. Decrease in CD93 (C1qRp) 71: 133–140. expression in a human monocyte-like cell line (U937) treated with various 4. Petrenko, O., A. Beavis, M. Klaine, R. Kittappa, I. Godin, and I. R. Lemischka. -inducing chemical substances. Microbiol. Immunol. 51: 1189–1200. 1999. The molecular characterization of the fetal stem cell marker AA4. Im- munity 10: 691–700. 23. Greenlee, M. C., S. A. Sullivan, and S. S. Bohlson. 2009. Detection and char- by guest on September 27, 2021 5. Norsworthy, P. J., L. Fossati-Jimack, J. Cortes-Hernandez, P. R. Taylor, acterization of soluble CD93 released during inflammation. Inflamm. Res. 58: A. E. Bygrave, R. D. Thompson, S. Nourshargh, M. J. Walport, and M. Botto. 909–919. 2004. Murine CD93 (C1qRp) contributes to the removal of apoptotic cells 24. Auwerx, J. 1991. The human leukemia cell line, THP-1: a multifacetted model in vivo but is not required for C1q-mediated enhancement of phagocytosis. J. for the study of monocyte-macrophage differentiation. Experientia 47: 22–31. Immunol. 172: 3406–3414. 25. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past: 6. McGreal, E. P., N. Ikewaki, H. Akatsu, B. P. Morgan, and P. Gasque. 2002. clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2: Human C1qRp is identical with CD93 and the mNI-11 antigen but does not bind 965–975. C1q. J. Immunol. 168: 5222–5232. 26. Kodama, T., M. Freeman, L. Rohrer, J. Zabrecky, P. Matsudaira, and M. Krieger. 7. Park, M., and A. J. Tenner. 2003. Cell surface expression of C1qRP/CD93 is 1990. Type I macrophage scavenger receptor contains alpha-helical and collagen- stabilized by O-glycosylation. J. Cell. Physiol. 196: 512–522. like coiled coils. Nature 343: 531–535. 8. Nepomuceno, R. R., S. Ruiz, M. Park, and A. J. Tenner. 1999. C1qRP is 27. Rohrer, L., M. Freeman, T. Kodama, M. Penman, and M. Krieger. 1990. Coiled- a heavily O-glycosylated cell surface protein involved in the regulation of coil fibrous domains mediate ligand binding by macrophage scavenger receptor phagocytic activity. J. Immunol. 162: 3583–3589. type II. Nature 343: 570–572. 9. Norsworthy, P. J., P. R. Taylor, M. J. Walport, and M. Botto. 1999. Cloning of the 28. Harhausen, D., V. Prinz, G. Ziegler, K. Gertz, M. Endres, H. Lehrach, P. Gasque, mouse homolog of the 126-kDa human C1q/MBL/SP-A receptor, C1qR(p). M. Botto, P. F. Stahel, U. Dirnagl, et al. 2010. CD93/AA4.1: a novel regulator of Mamm. Genome 10: 789–793. inflammation in murine focal cerebral ischemia. J. Immunol. 184: 6407–6417. Supplemental Figure 1.

AB ¢ ¤ 0JG+ 0Q 5GSWGPEG  ˧ Fc only NGCFGT (E  $7$**&7$*&*&&$&&$7***$7** £ ¥ :JQ+  7*7*7*$*7777*7&&*$*7**$&$7&7*7 ¢ § 0JG+ ©  $&$*$7*7&&$&7&**$&$$$$&7&$&$&$ hsCD93-Fc NGCFGT CC (E  &&$*&7&*$*&7$777$&&&**$*$&$* :JQ+ ¦ ¨ ¥  &*&&7&&*7*7&$*&&*$*7**$&$7&7*7  $&$*$7*7&&$&7&**&7*$&$&**$**&* ¢ § « 0JG+  7*7*7*$*7777*7&&&&&$7**7*&$$*$ hsCD93-Mucin-Fc NGCFGT CC (E  7&77*&$&&$7*****$&$$$$&7&$&$&$ ¦ ª ¥ :JQ+  7*7*7*$*7777*7&**&*7*$7**$7*&7 ¢ 0JG+  $*&$7&&$7&$&*&&*$&$$$$&7&$&$&$ hsCD93-His NGCFGT CC &7**&7&*$*&7$$7*$7*$7*$7*$7*$7*  ¬ Z*+5 :JQ+ &&&&$7**7*&$$*$

Supplemental Figure 1. (A) Schematic diagrams of three types of recombinant human CD93 and a control protein (Fc only). Each construct was amplified by overlap extension PCR, digested with Nhe I/ Xho I, and inserted to pcDNA3.1 vector. (B) List of primers used to construct various forms of recombinant human CD93 and control proteins. Supplemental Figure 2.

AB 300 ** **

200 (pg/mL)  100

TNF 130 95 %ORWDQWL KXPDQ)F 0 (E      

JU%&(E      

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.25    

Supplemental Figure 2. (A) THP-1 cells were cultured with 5 »g/ml of recombinant CD93 Fc fusion proteins, including hsCD93-Fc and hsCD93-Mucin-Fc, or controls for 48 h. Subsequently, cells were incubated with or without 1 »g/ml of LPS for an additional 24 h. Culture supernatants from THP-1 cells were collected and the concentration of TNFα was determined. Data represent the mean · SEM. (**, P < 0.01). Similar results were obtained from three independent experiments. (B) Comparison of expression levels between hsCD93-Fc and hsCD93-Mucin-Fc. Each construction vector was transfected and expressed on HEK293 cells. Culture supernatants were electrophoresed on 8% SDS-PAGE, transferred to nitrocellulose and blotted with anti-human Fc Ab. Supplemental Figure 3.

(-) Fc hIgG PMA 5 ug/ml 5 ug/ml 0.1 ug/ml

hsCD93-Fc hsCD93-Fc hsCD93-Fc hsCD93-Fc 5 ug/ml 1 ug/ml 0.2 ug/ml 0.04 ug/ml

Supplemental Figure 3. Phase-contrast images of THP-1 cells treated with negative controls (no treatment, Fc, or hIgG), a positive control (PMA), or various concentrations of hsCD93-Fc for 1 h. (Ý40 magnification) . Supplemental Figure 4.

(-) Fc only 1 μg/ml

hsCD93-Fc hsCD93-Fc 1 μg/ml 0.2 μg/ml

Supplemental Figure 4. Phase-contrast images of differentiated human monocytes treated with negative controls (no treatment, Fc, or hIgG) or hsCD93-Fc for 24 h. (×100 magnification). Supplemental Figure 5. 2JCUGEQPVTCUV 4(2 2JCUGEQPVTCUV 4(2

4(2 4(2

2JCUGEQPVTCUV 2JCUGEQPVTCUV

Supplemental Figure 5. Two representative photographic images of THP-1 cells that phagocytosed mRFP-expressing E. coli. THP-1 cells were differentiated for 3 days in the presence of 5 µg/ml of control hIgG (data not shown) or hsCD93-Fc and incubated for 6 h with the E. coli transformed by a mRFP expression vector. After washing with PBS, pictures were taken. Photomicrograph of hsCD93-Fc treated-THP-1 cells in visible merged with fluorescence, and photomicrograph with fluorescence only (RFP). Supplemental Figure 6.

Supplemental Figure 6. A gallery of representative confocal Z-stack images. THP-1 cells were differentiated for 3 days in the presence of 5 µg/ml of hsCD93-Fc and incubated for 6 h with E. coli transformed by a mRFP expression vector. After washing with PBS, cells were examined on a confocal laser scanning microscope (LSM 510; Carl Zeis Inc). A Z-stack of 40 images was aquired with a 2 µm interval. Images were analyzed by using the Zeis LSM software package. Supplemental Figure 7. 300 J

200 (pg/mL)  100 TNF

0 300 J 200 * (pg/mL)  100 TNF

0 300 ** J 200 (pg/mL)  100 TNF

0 (E       J+I)       JU%&(E       .25     Supplemental Figure 7. THP-1 cells were cultured with 5 »g/ml of hsCD93-Fc or controls for 12h, 24h, or 48 h. Subsequently, cells were incubated with or without 1 »g/ml of LPS for an additional 24 h. Culture supernatants from THP-1 cells were collected and the concentration of TNFα was determined with a Quantikine assay kit. Data represent the means · SEM. (**, P < 0.01; *, P < 0.05). Similar results were obtained from five independent experiments.