The Novel Lipopolysaccharide-Binding CRISPLD2 Is a Critical Serum Protein to Regulate Endotoxin Function

This information is current as Zhi-Qin Wang, Wen-Ming Xing, Hua-Hua Fan, Ke-Sheng of September 29, 2021. Wang, Hai-Kuo Zhang, Qin-Wan Wang, Jia Qi, Hong-Meng Yang, Jie Yang, Ya-Na Ren, Shu-Jian Cui, Xin Zhang, Feng Liu, Dao-Hong Lin, Wen-Hui Wang, Michael K. Hoffmann and Ze-Guang Han

J Immunol 2009; 183:6646-6656; Prepublished online 28 Downloaded from October 2009; doi: 10.4049/jimmunol.0802348 http://www.jimmunol.org/content/183/10/6646 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/10/28/jimmunol.080234 Material 8.DC1 References This article cites 46 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/183/10/6646.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Novel Lipopolysaccharide-Binding Protein CRISPLD2 Is a Critical Serum Protein to Regulate Endotoxin Function1

Zhi-Qin Wang,2* Wen-Ming Xing,2* Hua-Hua Fan,2† Ke-Sheng Wang,* Hai-Kuo Zhang,* Qin-Wan Wang,* Jia Qi,* Hong-Meng Yang,* Jie Yang,† Ya-Na Ren,‡ Shu-Jian Cui,* Xin Zhang,* Feng Liu,* Dao-Hong Lin,‡ Wen-Hui Wang,‡ Michael K. Hoffmann,§ and Ze-Guang Han3*

LPS is an immunostimulatory component of Gram-negative . Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a

novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood Downloaded from samples are 607 ␮g/ml and 290 ␮g/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2–0.9 ␮g/ml) and enhance CRISPLD2 (range, 1.5–4.2 ␮g/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-␣ and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations

increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum http://www.jimmunol.org/ concentrations not only are indicators of the degree of a body’s exposure to LPS but also reflect an individual’s LPS sensitivity. The Journal of Immunology, 2009, 183: 6646–6656.

lant and life evolved in a symbiotic relationship complex (6–11) that initiates the clonal expansion of immunocytes with the microbial world. Higher as well as hu- or the secretion of immunoregulatory cytokines (11). LPS-bind- P mans are exceedingly conscious of structural and func- ing reagents that down-regulate immunological LPS activities tional microbial components and entrust them with vital roles in have been reported (12–15). Of note is CD6, belong to the the regulation of their life functions (1, 2). Decades ago immunol- scavenger receptor cysteine-rich superfamily, which is readily ogists identified, in the cell wall of Gram-negative bacteria, a expressed on the surface of lymphocytes but maintains a low by guest on September 29, 2021 highly bioactive LPS composed of a monomorphic lipid core and serum concentration (16). It exhibits significant LPS-binding a polymorphic polysaccharide coat that stimulates immune func- affinity, and its soluble form has been shown to inhibit the in- tions in a systemic rather than local fashion (3). LPS-producing duction of endotoxic shock in mice (17). Passively administered Gram-negative bacteria pose a threat to the health of and LPS-reactive Abs have also been shown to ameliorate Gram- may kill them by septic shock (4, 5). negative sepsis (18–20). Mammals express an LPS-binding protein (LBP)4 that is con- In this study we present an LPS-binding molecule, cysteine- sidered a critical molecule in LPS-elicited activation cascades. rich secretory protein (CRISP) LCCL domain containing 2 LBP assembles upon reaction with LPS and several molecules on (CRISPLD2 and Crispld2, representing human and mouse/rat ver- the cell membrane, thus forming a molecular signal transduction sions, respectively), which was previously known for a variety of other functions. Because it is also known as late gestation 1 (Lgl1), it has been implicated in the development of rat lung (21– *Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese Na- tional Human Genome Center, Shanghai, China; †Department of Blood Engineering, 23) and mouse kidney (24) and is thought to be involved in the Shanghai Blood Center, Shanghai, China; and ‡Department of Pharmacology and development of nonsyndromic cleft lip with or without cleft palate §Department of Microbiology and Immunology, New York Medical College, Val- halla, NY 10595 (25). Its possession of two LCCL domains suggests a relationship to LPS. LCCL structures were initially described in the horseshoe Received for publication July 18, 2008. Accepted for publication September 19, 2009. crab (Limulus) factor C, which, by binding LPS, initiates the Limu- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance lus coagulation cascade to protect the crab against bacterial infec- with 18 U.S.C. Section 1734 solely to indicate this fact. tion (26–28). It seemed conceivable that mammalian Crispld2, 1 This study was supported by Chinese High-Tech Research and Development Program with two LCCL domains, exhibits particular avidity for LPS. Sev- Grants 2006AA02Z193 and 2006AA02A305, National Natural Science Foundation for eral known LPS-binding components of pulmonary surfactant, Outstanding Youth Grant 30425019, Chinese National Key Program on Basic Research Grant 2004CB518605, and the Shanghai Commission for Science and Technology. such as surfactant A and D, have been implicated in an 2 Z.-Q.W., W.-M.X., and H.-H.F. contributed equally to this work. Ab-independent host defense against pathogens (29, 30). Previ- ously, the analysis of leukocytes from healthy human volunteers 3 Address correspondence and reprint requests to Dr. Ze-Guang Han, Shanghai- MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China. E-mail address: [email protected] acid; ORF, open reading frame; RU, relative response unit; SPR, surface plasmon 4 Abbreviations used in this paper: LBP, LPS-binding protein; CHO, Chinese hamster resonance. ovary; CRISP, cysteine-rich secretory protein; CRISPLD2/Crispld2, CRISP LCCL domain containing 2 (human/mouse or rat); hTLR4, human TLR4; LTA, lipoteichoic Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802348 The Journal of Immunology 6647

i.v. administered bacterial endotoxin has shown the rapidly buffer without detergents by 140 V for 4 h, as compared with that of increased transcript of CRISPLD2 but not CRISPLD1 within 2 h rCRISPLD2 alone. The gels were stained with silver and scanned by Image (31), suggesting that transcription of CRISPLD2 in leukocytes is Scanner (GE Healthcare). immediately initiated in response to LPS challenge. The present Surface plasmon resonance (SPR) assay work introduces CRISPLD2 as a major serum protein To measure the affinity between CRISPLD2 and LPS, a SPR assay was that acts as a natural LPS antagonist and promises to be of con- performed to determine the recognition of LPS by CRISPLD2 with a Bia- siderable preventative value against endotoxic shock. core 3000 biosensor instrument (Biacore/GE Healthcare) using a C1 sensor chip (GE Healthcare). Briefly, either rCRISPLD2 or unrelated IgG at 0.18 Materials and Methods mg/ml diluted with acetate buffer (pH 4.5) was immobilized to the sensor chip by coupling with the primary amine group. Different concentrations of Bacterial components, mAbs, and rCRISP-3 protein E. coli LPS were injected into the flow cells at a rate of 20 ␮l/min. Ster- Escherichia coli (O127:B8 and O55:B5) LPS was purchased from Sigma- ilized and deaerated PBS was used as the running buffer during experi- Aldrich and dissolved in PBS at 1 mg/ml. Alexa Fluor 488-labeled E. coli ments. The sensorgram and relative response units (RU) for the binding of (O55:B5) and Salmonella enterica serovar Minnesota LPS was purchased LPS to immobilized CRISPLD2 were obtained by subtracting the back- from Invitrogen and dissolved in PBS at a final concentration of 1 mg/ml. ground of the LPS binding to immobilized, unrelated IgG. Following re- Purified Staphylococcus aureus lipoteichoic acid (LTA), ultra-pure moval of the unbound LPS by injection with PBS, the bound LPS was ␮ Rhodobacter sphaeroides LPS, and chimeric anti-human TLR4 (hTLR4)- removed by quick injection with 50 mM NaOH at a flow rate of 50 l/min. IgA mAbs were purchased from InvivoGen and dissolved in endotoxin- A saturation binding curve was depicted as a function of LPS concentra- free water according to the manufacturer’s instruction. Anti-hTLR4 tions vs RU. (HTA125) and OKT3 mAbs were purchased from eBioscienc. Human IgA LPS binding affinity analysis was purchased from Sigma-Aldrich. Recombinant CRISP-3 was purchased Downloaded from from R&D Systems. The dissociation constant of LPS (KD) was calculated by Scatchard plot analysis using the following formula: K ϭϪ1/slope ϭ (RU Ϫ RU)/ Plasmid constructs and cell transfection D max (RU/ConcLPS), where RUmax is maximum response and ConcLPS is the cDNA with an entire open reading frame (ORF) of human CRISPLD2 was concentration of LPS. In addition, KD can be calculated as the ratio of obtained from mixed tissues by PCR using the F1 forward primer 5Ј- these two constants (koff/kon). In brief, the dissociation rate constant koff is described by the equation R ϭ R eϪkoff(t Ϫ t0), and R is the amplitude of GCTGTCGCCGCTGCTACCGC-3Ј and the R1 reverse primer 5Ј- t t0 t0 the initial response. The association rate constant k can be derived from GACGCCCCTTCTCCCCTGGT-3Ј. The PCR product was inserted into on http://www.jimmunol.org/ ϭ Ϫ Ϫ(konC ϩ koff)(t Ϫ t0) the plasmid pGEM-T Easy Vector (Promega), which then served as the the measured koff value using: Rt Rmax [1 e ], where template in expression vector construction. To construct a recombinant C is the concentration of LPS, Rmax represents the maximum binding ca- prokaryote expression vector, partial human CRISPLD2 was inserted into pacity of LPS to immobilized CRISPLD2, and Rt represents the amount of pGEX-4T-1 (GE Healthcare) to generate the GST-CRISPLD2 (257–497 LPS bound to the CRISPLD2 at time t. The dissociation rate constant koff aa) fusion protein, which contains two LCCL domains and was used for is determined when the LPS passing over CRISPLD2 on the surface of chip generating the rabbit anti-CRISPLD2 polyclonal Ab. For mammalian cell is replaced by PBS alone. expression, human CRISPLD2 with the entire ORF was then subcloned Northern blot analysis and RT-PCR into pcDNA3.1A-myc-His (Invitrogen) to generate the c-myc-His-tagged CRISPLD2. The pcDNA3.1A-CRISPLD2 was transfected into CHO cells Northern blotting was performed by using a human multiple tissue North- by Lipofectamine (Invitrogen) and then selected with the antibiotic G418 ern blot membrane (Clontech) according to the manufacturer’s instruction. (Invitrogen). These stable CHO cells with human CRISPLD2 were main- The probe for detecting CRISPLD2 expression was amplified by PCR us- by guest on September 29, 2021 tained with G418, and many monoclonal recombinant CHO cell lines were ing the F2 forward primer 5Ј-TGGAATTCCGAGAAGAAACCTACA further screened out by limited cell dilution and Western blotting assays. CTC-3Ј and the R2 reverse primer 5Ј-TGCTCGAGATTCACTGCCTGAC AGCA-3Ј labeled with [␣-32P]dCTP using a random primer DNA labeling Anti-CRISPLD2 polyclonal antibody kit (Takara). Hybridization with a probe to human ␤-actin in the same The recombinant pGEX-4T-1 containing GST-CRISPLD2 (257–497 aa, membrane was used as a loading control. For RT-PCR, total RNA from human immune cells and mice tissues was extracted by TRIzol reagent with an LCCL domain) was transformed into E. coli BL21 for prokaryote ␮ expression. The GST-CRISPLD2 expression in bacteria was induced by (Invitrogen). Reverse transcription was performed in 20- l reactions using 2 ␮g of total RNA. Each PCR was generally performed with ϳ30–32 isopropyl-␤-D-thiogalactopyranoside at 28°C, followed by centrifugation at 8,000 ϫ g at 4°C for 10 min. The supernatant was discarded and the pellet thermal cycles, and the PCR products were detected by electrophoresis on ␮ a 2% agarose gel with ␤-actin used as a loading control. The PCR primers was resuspended in PBS with 5 mM DTT and 100 g/ml PMSF. After Ј sonication, the lysate was centrifuged at 8,000 ϫ g again at 4°C for 10 min. used in this study for Crispld2 (mouse) were 5 -GCGGTCGACGCCAT GAGCTGCGTCCTG-3Ј (forward) and 5Ј-GCGCTCGAGTCACTGCCT The GST-CRISPLD2 (257–497 aa) in the supernatant was purified using Ј an affinity column of glutathione-Sepharose 4B (GE Healthcare). Normal GACAGCAAAGATC-3 (reverse), and the primers used for CRISPLD2 (human) were 5Ј-AGT/CTAGACATGAGCTGCGT-3Ј (forward) and 5Ј- rabbits were immunized with the purified protein and the polyclonal Abs Ј were purified from the sera using protein G-Sepharose (GE Healthcare). GGGAATTCTCTGCCTGACAG-3 (reverse). The PCR primers used for human TLR4 were 5Ј-CTGCAATGGATCAAGGACCA-3Ј (forward) and Purification of rCRISPLD2 5Ј-TCCCACTCCAGGTAAGTGTT-3Ј (reverse), those used for MD2 were 5Ј-TTCCACCCTGTTTTCTTCCA-3Ј (forward) and 5Ј- TCATCA The serum-free cell medium of CHO cells with stably transfected GATCCTCGGCAAAT-3Ј) (reverse), and the PCR primers used for ␤-ac- pcDNA3.1A containing human CRISPLD2 was collected. The rCRISPLD2 in tin were 5Ј-TGAACCCTAAGGCCAACCGTG-3Ј (forward) and 5Ј-GCTC the supernatant was denaturalized by 4 M urea and purified by Ni-NTA ATAGCTCTTCTCCAGGG-3Ј (reverse). metal-affinity chromatography (Qiagen). After two washes with 50 ␮lof 2 washing buffer (50 mM Na HPO4, 300 mM NaCl, and 4 M urea; (pH 7.5)), Isolation of human immune cells and flow cytometry analyses the purified protein was eluted in buffer with 250 mM imidazole (pH 7.8). The collected fractions were then pooled and dialyzed against PBS. SDS- Human peripheral blood was donated by healthy volunteers and PBMCs PAGE was used to evaluate the purity of CRISPLD2-myc-His fusion by were isolated by Ficoll (Pharmacia/GE Healthcare) density gradient cen- Coomassie blue staining. The BCA assay kit (Pierce) was used to deter- trifugation. After whole blood was collected in acidic citrate dextrose (1:5 mine protein concentration. (v/v) blood) and centrifuged at room temperature to form a leukocyte layer, granulocytes were precipitated by Ficoll (Pharmacia/GE Healthcare) den- Native PAGE mobility shift assay sity gradient centrifugation from the resuspension of leukocytes layer, and then remaining erythrocytes were removed by hypotonic lysis. Generally, To evaluate the interaction of CRISPLD2 with LPS, a native PAGE mobility T cells, NK cells, and monocytes were isolated from human PBMCs by shift assay was used as described (7). Briefly, the purified rCRISPLD2 protein negative selection using specific Ab-coated magnetic beads (Dynabeads) was incubated with either E. coli or S. enterica serovar Minnesota LPS at according to manufacturer’s instructions (Invitrogen). To further confirm 37°C for 30 min. Bromophenol blue and glycerol were loaded into samples the up-regulation of CRISPLD2 secretion from purified human T cells by and electrophoresis was preformed on 10% native polyacrylamide gel to activation of TLR4, CD4 T cells (95% purity) were isolated twice by a measure the mobility of the mixtures with different LPS doses in a running CD4 T cell negative selection kit (Miltenyi Biotec). For RT-PCR analysis 6648 SERUM LIPOPOLYSACCHARIDE-BINDING PROTEIN CRISPLD2

ϩ of TLR4 transcript in T cells, the CD4 T cells (98% purity) were first medium containing 0.1% FCS, and incubated in 5% CO2 at 37°C. To isolated by CD4 T cells negative selection kit and then, in second step, determine the reduction by CRISPLD2 of LPS-induced TNF-␣ and IL-6, purified by a CD4 T cell positive selection kit. CD14ϩ cells, as a positive PBMCs at 1 ϫ 106 cells/ml were incubated in the presence of multiple control for TLR4 transcript, were purified by a CD14 monocyte positive concentrations of rCRISPLD2 or rCRISP-3 in RPMI 1640 medium with ␮ selection kit (Miltenyi Biotec). To assess CRISPLD2 on cell surfaces, PBMCs 0.3% FCS in 5% CO2 at 37°C and stimulated by LPS (10 g/ml). To assess (106 cells/ml) in PBS with 1% inactivated human serum were seeded into the regulatory function of endogenous and exogenous CRISPLD2, human ␮ 96-well plates and incubated with 15 g/ml rabbit anti-CRISPLD2 poly- PBMCs were incubated in RPMI 1640 with 0.1% FCS in 5% CO2 at 37°C, clonal Ab for 30 min. After washing three times with buffer, the cells were and the other supplement regents, including human serum, rCRISPLD2, E. stained with Cy2-labeled goat anti-rabbit IgG for 30 min. Normal rabbit coli LPS, anti-CRISPLD2 Ab, and control Ab, were used. IgG was used as a negative control. To determine intracellular CRISPLD2, immunofluorescence assays were used to simultaneously detect surface ELISA cluster of differentiation markers and intracellular CRISPLD2 of these Commercial TNF-␣ and IL-6 ELISA kits (R&D Systems) were used to cells. PBMCs were seeded into 96-well plates, stained with PE-labeled measure human cytokines in culture supernatants and serum according to (red) anti-CD4, CD8, CD56, or CD14 mAb (BD Biosciences) in the pres- manufacturer’s instruction. For measuring CRISPLD2/Crispld2 in culture ence of 2% inactivated human serum, and fixed with 4% paraformaldehyde supernatants and mouse serum, appropriately diluted samples in sodium in PBS. After washing twice with Perm/Wash buffer (BD Pharmingen), the carbonate buffer (pH 9.5)-coated 96-well microtiter plates (Nunc Maxi- cells were incubated with either rabbit anti-CRISPLD2 polyclonal Ab or Sorp) overnight at 4°C. After washing twice with PBS, the plates were first ␮ normal rabbit IgG for at 15 g/ml for 1 h. The cells were washed three incubated with 8% FCS in PBS for 60 min and subsequently with rabbit ϫ times with 1 Perm/Wash buffer again and stained by Cy2-labeled (green) anti-CRISPLD2 Ab (10 ␮g/ml) in PBS with 8% FCS for 2 h. After washing goat anti-rabbit IgG for 30 min. T cell-, NK cell-, and monocyte-specific four times, HRP-conjugated donkey anti-rabbit IgG Ab (Jackson Immuno- surface markers and the intracellular CRISPLD2 were detected by a Research Laboratories) was added. The tetramethylbenzidine substrate in FACSCalibur flow cytometer (BD Biosciences). T cells, NK cells, and mono- sodium acetate/citric acid buffer (0.1 M; pH 6.0) was used for visualization. cytes were gated according to their characteristic surface marker vs forward The catalytic action was stopped by 2.0 M sulfuric acid. OD was measured in Downloaded from scatter characteristics. Granulocytes were gated according to their typical for- a spectrophotometer at 450 nm with the correction wavelength set at 570 nm. ward vs side scatter characteristics. A standard curve of rCRISPLD2 titration (2.0, 1.0, 0.5, 0.25, 0.125, 0.062, ␮ Immunoblotting analysis 0.031,and 0 g/ml) was used for the quantification of CRISPLD2 and for the reference of mouse Crispld2. It should be pointed out that the concentration of The samples of serum and plasma from human volunteers and mice were bovine Crispld2 in FCS is very low, and the incubation with 8% FCS does not diluted 1/10 in PBS, separated by electrophoresis in 10% SDS-PAGE gels significantly increase the background in ELISA (see supplemental Fig. S7).5

and then transferred onto nitrocellulose membranes (GE Healthcare). The For measuring anti-LPS Ab in mouse serum, LPS (100 ng/ml) in sodium http://www.jimmunol.org/ references of rCRISPLD2 in PBS (0.3, 0.1, 0.03, and 0.01 mg/ml) were carbonate buffer (pH 9.5) was plated in 96-well microtiter plates overnight, used as control for diluted samples. The electrophoresis was performed in followed by treatment with 10% FCS in PBS for 60 min. Mouse sera in 1- to loading buffer without DTT for retaining IgG above the 150 kDa position 100-fold dilutions in PBS were added to the immobilized LPS, and HRP- on a gel to avoid overlap of the IgG H chain with CRISPLD2/Crispld2. The conjugated donkey anti-mouse Ab (Jackson ImmunoResearch Laboratories) membranes were blocked with 5% dry skim milk and incubated with rabbit was used to react with plate-bound anti-LPS Abs. anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab Mice and in vivo LPS stimulation coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting mem- branes were finally scanned by the Odyssey infrared imaging system (LI- Six- to 8-wk-old BALB/c female mice were purchased from SIPPR-BK COR Biosciences). The serum CRISPLD2 concentration, represented by Experimental Animal Ltd. All mice were housed in the animal facility of the density of each spot, was calculated according to reference of the

Shanghai Jiao Tong University Medical School (Shanghai, China). All pro- by guest on September 29, 2021 rCRISPLD2 standard. cedures with mice were performed according to guidelines of the Harvard Medical School Office for Research Subject Protection (Boston, MA) and Immunofluorescence assay and immunohistochemical staining approved by the ethics committee of the Chinese National Genome Center For identification of Crispld2 in mouse and rat tissues, freshly sacrificed (Shanghai, China). To determine serum Crispld2 concentration in response mice and rats were arterially perfused with 4% paraformaldehyde in PBS. to LPS and LTA, 6- to 8-wk-old BALB/c mice were injected i.p. with S. The excised tissues were fixed in the same buffer. Paraffin-embedded tissue aureus LTA (0.1 mg/mouse) and E. coli-type LPS (0.015, 0.030, and 0.10 sections (6 ␮m) were mounted on glass slides, the sections were deparaf- mg/mouse), respectively, and blood samples were taken from the tail vein finized and rehydrated and the mouse placenta tissue was excised and fro- on days 0, 1, 2, 5, 7, 12, and 31. For the prevention of LPS-induced en- zen then sectioned with a cryostat at 6 ␮m. All tissue sections on glass dotoxin shock, 6- to 8-wk-old BALB/c female mice were injected i.p. with slides were stained for 60 min with anti-CRISPLD Ab diluted in PBS E. coli (O55:B5) LPS (0.45 mg/mouse; 22.5 mg/kg) in the absence or containing 1% Tween 20. For immunofluorescent staining, Cy2-labeled presence of recombinant human CRISPLD2 (molecular mass, 55 kDa; 1.4 second Ab was used for recognition of the primary Ab. Hoechst dye was mg/mouse; 70 mg/kg) and CRISP-3 (molecular mass, 25 kDa; 0.64 mg/ used for nuclear staining. Immunofluorescence was visualized using a con- mouse; 32 mg/kg), respectively. To monitor the serum CRISPLD2 re- focal microscope (LSM 510, Version 3.5 META; Zeiss). For immuno- sponse to repeated LPS challenges, 6- to 8-wk-old BALB/c female mice chemical staining, the HRP-conjugated anti-rabbit secondary Ab (Dako- were first injected i.p. with E. coli (055:B5) LPS (0.03 mg/mouse; 1.5 Cytomation) was used as the secondary Ab, and the signals were detected mg/kg). After 10 days, these mice were separated into four groups and using a diaminobenzidine substrate kit (Vector Laboratories). Nuclear again injected i.p. with LPS at 25, 40, 55, and 70 mg/kg, respectively. After staining was conducted by hematoxylin. 32 days, surviving mice received a third injection of LPS (45, 60, or 75 mg/kg). Serum Crispld2 and anti-LPS Ab were examined before i.p. in- LPS binding to immune cells jection of high doses of LPS. PBMCs were seeded into 96-well plate at 2 ϫ 106 cells/ml in PBS con- Statistics analyses taining 0.3% BSA with different doses of rCRISPLD2 or rCRISP-3 (0, Student’s t test was used for the comparison of two independent groups. 0.018, 0.054, 0.178, 0.535, 1.78, and 5.35 ␮M) in the presence of Alexa Statistical difference of survival rates between two groups with endotoxin Fluor 488-labeled E. coli LPS (1.0 and 3.0 ␮g/ml) or S. enterica serovar shock was analyzed by a Kaplan-Meyer log-rank test. Statistic analyses on Minnesota LPS (9.0 ␮g/ml) for 30 min. After washing twice, the mean the correlation between serum Crispld2 concentrations vs lethal LPS dose fluorescent intensity of Alexa Fluor 488 on monocytes and lymphocytes were performed by GraphPad Prism 5 software. For all tests, a value of p Ͻ was examined by a FACSCalibur flow cytometer (BD Biosciences). Mono- 0.05 was considered statistically significant. cyte and lymphocyte regions were distinguished according to their forward vs side scatter characteristics. The fluorescence signal was represented by mean fluorescence intensity units. Results Molecular analysis and LPS reactivity Stimulation of immune cells in vitro To examine the possibility that CDRISPLD2 is a LPS-binding pro- For assessing CRISPLD2 secretion from cells in vitro, human granulocytes tein, we isolated the whole ORF of human CRISPLD2 and stably at 1 ϫ 106 cells/ml, PBMCs, purified T cells, and NK cells at 2 ϫ 106 cells/ml, and purified monocytes at 0.5 ϫ 106 cells/ml were seeded into 96-well plates, stimulated by LPS and LTA, respectively, in RPMI 1640 5 The online version of this article contains supplemental material. The Journal of Immunology 6649

Table I. Affinity of LPS to its receptors and soluble binding proteins

a Immobilized LPS KD (M) Refs. CRISPLD2 2.46 Ϯ 0.8 ϫ 10Ϫ6 Fig. 1D CRISPLD2 1.33 Ϯ 0.9 ϫ 10Ϫ6 Fig. 1Bb LBP 1.40 ϫ 10Ϫ8 8 CD14 0.98 ϫ 10Ϫ5 8 CD14 0.87 Ϯ 0.2 ϫ 10Ϫ5 32 MD-2 2.33 Ϯ 0.9 ϫ 10Ϫ6 32 TLR4 1.41 Ϯ 0.7 ϫ 10Ϫ5 32

a All KD values were measured by SPR assay. b Data from three independent experiments (mean Ϯ SD).

rized in Table I, indicating that the affinity of CRISPLD2 for LPS is similar to that for CD14 and MD2 but lower than that for LBP (8, 32). To assess the molecular phylogeny of CRISPLD2, we also com-

pared CRISPLD2 with homologous protein sequences deposited in Downloaded from GenBank by using bioinformatic tools. We found that CRISPLD2 is highly conserved throughout evolution (supplemental Fig. S2).

Tissue expression and serum concentration RT-PCR technology was applied to demonstrate the occurrence of

Crispld2 mRNA in six mouse tissues (supplemental Fig. S3A). In http://www.jimmunol.org/ addition, Crispld2 was immunofluorescently identified in tissue FIGURE 1. CRISPLD2 binds LPS but not S. aureus LTA. A, Reaction sections of mouse lung, placenta, and small intestine (supplemen- with S. enterica serovar Minnesota (S. minnesota) and E. coli LPS shifts tal Fig. S3B), and also immunohistochemically identified in rat the mobility of rCRISPLD2 in a native PAGE mobility shift assay. B and lung and intestine (supplemental Fig. S3C). Northern blot analysis C, Sensorgrams of E. coli LPS (B) and S. aureus LTA (C) binding to revealed that the CRISPDL2 gene is transcribed in leukocytes and immobilized CRISPLD2. Either E. coli LPS or S. aureus LTA (0.3, 1.0, many human organs; it is most pronounced in the placenta and also ␮ 3.0, 10, and 30 M) was passed over immobilized CRISPLD2. The dis- in heart, lung, small intestine, and blood leukocytes, but far less sociation constant of LPS, K , was calculated as the ratio of these two D pronounced in liver, kidney, spleen, thymus, colon, muscle, and constants (k /k ) according to the binding parameters from a sensor- off on by guest on September 29, 2021 gram. D, RU that reflects E. coli LPS binding activity was measured by brain (supplemental Fig. S3D). a BIAcore instrument when E. coli LPS (1.0, 3.0, 10, 30, and 100 ␮M) CRISPLD2 was identified and quantified in healthy human se- was passed over the CRISPLD2 chip. E, Scatchard plot analysis and the rum (range, 384–790 ␮g/ml; n ϭ 7) and plasma (range, 402–998 Ϯ ␮ ϭ KD of E. coli LPS binding to CRISPLD2 (mean SD). g/ml; n 3) by using an immunoblotting assay (Fig. 2A). How- ever, the concentrations of CRISPLD2 in infant umbilical cord blood plasma (range, 149–426 ␮g/ml; n ϭ 5) are lower than those transfected it into CHO cells using the mammalian expression in adult blood plasma (Fig. 2B). As compared with published se- plasmid pcDNA3.1 for expressing the recombinant c-myc-His rum concentrations of known LBPs, the serum levels of tagged CRISPLD2. rCRISPLD2 was purified from culture me- CRISPLD2 are substantially higher than those of LBP, soluble dium and evaluated by electrophoresis on SDS-polyacrylamide CD14, bactericidal/permeability-increasing protein, and soluble gels and by immunoblotting assays using c-Myc Abs and home- CD6 (supplemental Table I, Fig. 2, A and B, and Refs. 16, 33, and made anti-CRISPLD2, respectively (supplemental Fig. S1). To as- 34). Moreover, human peripheral blood granulocytes and mono- sess potential CRISPLD2 LPS-binding activity, we used an EMSA nuclear cells, including monocytes, NK cells and T cells, express on native polyacrylamide gel using purified rCRISPLD2 together CRISPLD2 that was identified by RT-PCR (Fig. 2C) in concor- with LPS from E. coli or S. enterica serovar Minnesota. The elec- dance with published microarray data for gene expression (35–38). trophoretic mobility of the CRISPLD2 band was significantly CRISPLD2 was readily seen in the cytoplasm of PBMCs (Fig. 2, shifted by E. coli and S. enterica serovar Minnesota LPS (Fig. 1A), D and F), including monocytes, NK cells, and T cells (supplemen- indicating that CRISPLD2 did interact physically with LPS. To tal Fig. S4F), by using intracellular Ab staining, but we were un- further confirm the finding, we performed a SPR assay by using a able to detect it on the cell surface by using extracellular Ab stain- BIAcore 3000 biosensor to quantitatively assess the recognition of ing (Fig. 2E). Interestingly, intracellular Crispld2 was also found E. coli LPS by CRISPLD2; S. aureus LTA was used as a control. in lamina propria lymphocytes in the small intestines of mouse and The sensorgrams showed that E. coli LPS, but not S. aureus LTA, rat (supplemental Fig. S3, B and C). binds to immobilized CRISPLD2 (Fig. 1, B and C). The relative RU of LPS binding to immobilized CRISPLD2 was measured by Soluble rCRISPLD2 interferes with LPS target cell binding subtracting a background of LPS binding to immobilized, unre- The ability of soluble rCRISPLD2 to intercede in the interaction of lated IgG. The binding of E. coli LPS to immobilized CRISPLD2 LPS with its target cells was tested under several experimental occurs in a dose-dependent manner (Fig. 1D). Therefore, we ten- conditions. Fig. 3, A and B, revealed that the presence of Ϯ ␮ tatively assessed an apparent KD that is 2.46 0.84 M for the CRISPDL2, but not CRISP-3, in the culture medium inhibited the binding of LPS to CRISPLD2 (Fig. 1E). The LPS affinity to reaction of two LPS preparations with cellular LPS receptors on CRISPLD2 was compared with LPS affinity to known soluble monocytes in a dose-dependent fashion. Accordingly, the release binding proteins and receptors by using an SPR assay as summa- of the cytokines TNF-␣ (Fig. 3C) and IL-6 (Fig. 3D) subsided in 6650 SERUM LIPOPOLYSACCHARIDE-BINDING PROTEIN CRISPLD2 Downloaded from

FIGURE 2. CRISPLD2 occurs in cells and in serum. A, Immunoblots (IB) of 1/10 diluted sera from healthy volunteers (right; n ϭ 7) and the rCRISPLD2 as reference (left) were used for assessing the quantity of the protein in human sera. B, Comparison of CRISPLD2 plasma levels in the blood of healthy adults (n ϭ 3) with the levels infant umbilical cord blood (n ϭ 5). The density of spots on film was scanned and calculated by using an Odyssey infrared imaging system. No DTT was in the loading buffer. Recombinant human CRISPLD2 served as a reference standard. C, CRISPLD2 mRNA from sub- populations of human white blood cells, including granulocytes, PBMCs, monocytes, T cells, and NK cells was subjected to RT-PCR. ␤-Actin served as http://www.jimmunol.org/ the loading control. RNA extracted from CHO cell line with stable CRISPLD2 transfection was used as positive control (ϩ), and clean water was used as a negative control (Ϫ). D, Confocal microscope photographs of human PBMCs fixed with 4% paraformaldehyde, permeated with 0.1% Triton, and stained with anti-CRISPLD2 Ab as compared with normal rabbit IgG; goat anti-rabbit IgG-Cy2 Ab was used as a secondary Ab (red). The photographs are in 200-fold (panels 1 and 3) and 1000-fold (panels 2 and 4) magnification. E, PBMCs were directly stained with rabbit anti-CRISPLD2 Ab (open area) or a control Ab (shaded areas) in PBS. F, PBMCs were permeabilized by Perm/Wash buffer and stained with rabbit anti-CRISPLD2 Ab (open area) or control Ab (shaded areas) in Perm/Wash buffer; Cy2-labled anti-rabbit IgG Ab served as a secondary Ab. A fluorescent signal was only obtained in the cytoplasm and not on the surface of cells. by guest on September 29, 2021 the presence of the LPS-binding molecule but not in the presence into the culture medium, but quite substantial amounts accumu- of CRISP-3, a cysteine-rich secretory protein without LCCL do- lated in the presence of LPS (Fig. 4, A–E). It is possible that LPS main (Fig. 3, C and D). up-regulates CRISPLD2 release in such a short time (Fig. 4, A–E) TLR4 has been found on surface of NK cells (39). Fluorescence- due to constant transcription and translation of CRISPLD2 in the labeled E. coli and S. enterica serovar Minnesota LPS were de- these cells (Fig. 2, C–F, and supplemental Fig. S4F). The up- tectable on CD56ϩ cells and CD56Ϫ lymphocytes in our experi- regulation of CRISPLD2 was observed not only in leukocyte cul- ment (supplemental Fig. S4, D and E), although the LPS ture but also in animals. The Crispld2 levels rose substantially in fluorescence intensity on these cells was far lower than that on the serum of LPS-treated mice and seemed to remain higher over monocytes (supplemental Fig. S4, A–E). Expectedly, rCRISPLD2 1 wk. Fig. 4G shows immunoblot data obtained with sera from prevented LPS binding to CD56ϩ cells and CD56Ϫ lymphocytes mice taken several days after treatment with nontoxic doses of (supplemental Fig. S4, D and E). LPS. A similar picture presents itself in an ELISA of Crspld2 in To determine whether CRISPLD2 is capable of dislodging LPS such sera (Fig. 4F). after it has reacted with cellular surface receptors, PBMCs were Rhodobacter sphaeroides LPS is the potent antagonist of toxic incubated with labeled LPS for 30 min before unbound LPS was LPS in both human and murine cells and prevents LPS-induced removed and CRISPLD2 was added. Fig. 3, E and F, suggest that shock in mice (40). In the present study we found that ultra-pure in sufficient doses, competitive removal of bound LPS from mono- R. sphaeroides LPS alone up-regulated CRISPLD2 secretion and cyte surfaces occurs in the presence of CRISPLD2. Fig. 3G re- induced significantly lower TNF-␣ production (Table II). To de- vealed that LPS-induced TNF-␣ production was significantly al- termine whether the LPS-induced up-regulation of CRISPLD2 se- tered even when CRISPLD2 was added after LPS. However, the cretion could be blocked by a neutralizing mAb against TLR4, we reduced CRISPLD2 inhibitory activity at 6 h after LPS may reflect used an anti-TLR4 (HTA125) mAb and a humanized chimeric a prior commitment of target cells to LPS response rather than anti-hTLR4-IgA mAb in vitro experiments. According to the ref- consolidation of LPS binding. erence data from the manufacturer (InvivoGen), the efficacy of the humanized chimeric IgA mAb is 100-fold higher than that of the LPS and an anti-hTLR4-IgA mAb up-regulate CRISPLD2 HTA125 mAb for blocking LPS-induced intracellular activation. secretion In concordance with the reference data, the anti-hTLR4-IgA mAb In the course of these experiments, it was noted that not only does effectively suppressed E. coli LPS-induced TNF-␣ and IL-6 pro- CRISPDL2 curtail LPS bioregulatory function, but also in turn duction in our experimental system (Table II). Unexpectedly, both LPS up-regulates the release of CRISPLD2 by granulocytes and anti-TLR4 mAbs were failed to block E. coli LPS-induced up- PBMCs, including purified monocytes, T cells, and NK cells. regulation of CRISPLD2 secretion. Interestingly, we found that the Spontaneously, the cells emitted lower quantities of CRISPLD2 anti-hTLR4-IgA mAb alone, but not anti-TLR4 (HTA125) mAb or The Journal of Immunology 6651

FIGURE 3. CRISPLD2 prevents the binding of LPS to target cells and inhibits the release of proinflamma- tory factors. A, Soluble rCRIPLD2 but not rCRISP-3 prevents Alexa Fluor 488-labeled E. coli LPS (1.0 ␮g/ ml) binding to human PBMCs. Monocytes and lympho- cytes were gated respectively, and mean fluorescence intensity (MFI) was determined by using flow cytom- etry analysis. Open symbols represent lymphocytes, and closed symbols represent monocytes. The absence of LPS (No LPS) was used as a negative control. B, The same experimental system was used with Alexa Fluor

488-labeled S. enterica serovar Minnesota (S. minne- Downloaded from sota) LPS (9.0 ␮g/ml). C and D, Soluble rCRISPLD2, but not rCRISP-3, inhibits E. coli (Ec) LPS (10 ng/ml)- induced TNF-␣ (C) and IL-6 (D) release from human PBMCs. Open symbols represent the absence of LPS stimulation used as a negative control, and closed sym- bols represent the presence of E. coli LPS. E and F, Soluble rCRISPLD2 dislodges cell-bound LPS from http://www.jimmunol.org/ monocyte surfaces. PBMCs were incubated with Alexa Fluor 488-labeled E. coli (3.0 ␮g/ml) or S. enterica se- rovar Minnesota (S. m) (9.0 ␮g/ml) LPS for 30 min before unbound LPS was removed and rCRISPLD2 was added. After incubation with rCRISPLD2 for 30 min at 4°C, the bound LPS on monocytes (closed symbols) and lymphocytes (open symbols) were analyzed by using flow cytometry. The absence of LPS (No LPS) was used

as a negative control. G, Recombinant CRISPLD2 in- by guest on September 29, 2021 terferes with LPS-induced cytokine production even when added several hours after LPS induction. Human PBMCs were stimulated with E. coli LPS (100 ng/ml) and rCRISPLD2 was added at the indicated time points. Cytokines released in the culture supernatants at 18 h were measured by ELISA, and the absence of rCRISPLD2 (Ϫ) was used as a positive control.

control human IgA, was capable of up-regulating CRISPLD2 se- of purified S. aureus LTA-treated mice (100 ␮g/per-mouse) did cretion (Table II and supplemental Fig. S5A). Moreover, the chi- not significantly rise (Fig. 4F), implying that S. aureus LTA bind- meric anti-hTLR4-IgA mAb synergized the E. coli LPS-induced ing to CD6 and S. aureus LTA-induced activation of TLR2 is up-regulation of CRISPLD2 release (Table II). Together, the re- irrelevant to the up-regulation of CRISPLD2 release. Whether the sults in Table II suggested that the up-regulation of CRISPLD2 activation of other TLRs, except TLR4 and TLR2, up-regulate secretion can be triggered by TLR4-mediated signaling that seems CRISPLD2 release is to be further investigated. to be irrelevant to TNF-␣ production. To determine whether the TLR4/MD2 receptor complex is It is known that purified S. aureus LTA is a ligand of CD6 (17) expressed on T lymphocytes, RT-PCR was applied to demon- that can activate TLR2 but no other TLRs, including TLR4 (41). strate the occurrence of TLR4 and MD2 mRNA in PBMCs, We investigated whether purified S. aureus LTA up-regulates purified CD4ϩ T cells (98% pure), and CD14ϩ monocytes (96% CRISPLD2 release in vivo and in vitro. Fig. 4, A and B, and sup- pure), and PE-labeled anti-TLR4 mAb was also used to deter- plemental Fig. S5B show that purified S. aureus LTA (1 ␮g/ml) mine whether TLR4 anchors on the surfaces of CD4ϩ and failed to up-regulate CRISPLD2 release from PBMCs, purified CD8ϩ T cells and CD14 monocytes in PBMCs. Supplemental CD4 T cells, and monocytes. Furthermore, Crispld2 serum levels Fig S4G revealed that TLR4 and MD2 transcripts were detected 6652 SERUM LIPOPOLYSACCHARIDE-BINDING PROTEIN CRISPLD2

FIGURE 4. LPS promotes human granulocytes and PBMCs to secrete CRISPLD2 and increases the CRISPLD2 serum levels. A and B, Human PBMCs (A) and monocytes (B) were stimulated with E. coli LPS and S. aureus LTA, respectively, and CRISPLD2 in supernatants were quantitated at various time points. C–E, Human granulocytes (C), NK cells (D), and T cells (E) were stim- Downloaded from ulated with or without E. coli LPS, and the CRISPLD2 concentrations in culture medium were quantitated by ELISA (mean of three experiments Ϯ SD). F and G, Up-regulation of Crispld2 expression in mouse serum after administration of a nontoxic http://www.jimmunol.org/ dose of LPS. BALB/c mice were in- jected i.p. with S. aureus LTA (100 ␮g/mouse) and E. coli type LPS (15, 30 and 100 ␮g/mouse) respectively. F, Western blotting (IB, immunoblot- ting) was used to determine Crispld2 in mice sera (E. coli LPS 100 ␮g/ mouse) at days (D) 0, 1, 2, 5 and 7.

The mice sera were diluted 1/10 in by guest on September 29, 2021 PBS for PAGE separation, so real concentrations of Crispld2 are ϳ10 times higher. G, Serum Crispld2 from mice treated with S. aureus LTA (100 ␮g/mouse) or E. coli LPS (15, 30 and 100 ␮g/mouse) was measured by ELISA at different time points. Re- combinant human CRISPLD2 was used as reference for western blot and ELISA.

in CD4ϩ T cells as compared with CD14 monocytes and PBMCs. To exclude the possibility that the up-regulation of CRISPLD2 Moreover, supplemental Fig S4H demonstrated that TLR4 was found secretion from T cells is a secondary response or side effect of on the cell surfaces of CD4 and CD8 T lymphocytes via immunoflu- contamination that could stimulate T lymphocytes, in the present orescence assay, which is consistent with the published literature work CD4ϩ T cells from PBMCs were purified twice by negative (42–44). To further confirm LPS binding to TLR4 on T cells, the selection to reach high purity (95%), and the anti-hTLR4-IgA mAb anti-hTLR4-IgA mAb was used to interfere with the binding of FITC- was extensively dialyzed to exclude possible contamination. Also, labeled E. coli LPS to PBMCs stained with PE-conjugated anti-CD4 a stimulatory anti-CD3 mAb (OKT3) was used to mimic contam- and CD8 mAbs, respectively. The results revealed that anti-hTLR4- ination for activating the TCR/CD3 complex. The results showed IgA mAb, but not control IgA, partially blocked the binding of E. coli that the stimulation of anti-hTLR4-IgA mAb, but not pure S. au- LPS-FITC to CD4ϩ and CD8ϩ T lymphocytes (supplemental Fig. reus LTA or human IgA, effectively increased the CRISPLD2 se- S4I), suggesting that LPS can bind to TLR4 on the surface of T cells. cretion from purified CD4 T cells in a short time, as compared with The Journal of Immunology 6653

Table II. Both R. sphaeroides LPS and anti-hTLR4-IgA Ab up-regulate CRISPLD2 secretion and reduce E. coli LPS-induced TNF-␣ and IL-6 production

b Concentration CRISPLD2b TNF-␣ IL-6b Treatmenta (␮g/ml) (␮g/ml) (ng/ml) (ng/ml)

Controlc 0.37 Ϯ 0.10 0.04 Ϯ 0.01 0.11 Ϯ 0.02 E. coli LPS 1 1.59 Ϯ 0.26 8.56 Ϯ 0.62 10.5 Ϯ 0.28 R. sphaeroides LPS 1 1.40 Ϯ 0.06 3.45 Ϯ 0.27d 7.98 Ϯ 0.22d E. coli LPS ϩ R. sphaeroides LPS 1 ϩ 1 1.65 Ϯ 0.09 3.29 Ϯ 0.07d 7.25 Ϯ 0.59d Anti-hTLR4-IgA 1 1.66 Ϯ 0.04 0.08 Ϯ 0.03 0.11 Ϯ 0.03 Anti-hTLR4-IgA 5 1.93 Ϯ 0.21 0.13 Ϯ 0.01 0.07 Ϯ 0.03 E. coli LPS ϩ anti-hTLR4-IgA 1 ϩ 1 2.25 Ϯ 0.24e 3.17 Ϯ 0.09d 7.06 Ϯ 0.45d E. coli LPS ϩ anti-hTLR4-IgA 1 ϩ 5 2.55 Ϯ 0.43e 2.63 Ϯ 0.07d 5.27 Ϯ 0.68d

a Human PBMCs were treated by the reagents as indicated in table for 12 h. b CRISPLD2, TNF-␣, and IL-6 were measured by ELISA in culture supernatants. c Ten microliters of PBS was added in culture as control. Data (mean Ϯ SD of triplicate wells) are from two independent experiments. d Value of p Ͻ 0.005 as compared with that from E. coli LPS treatment alone. e Value p Ͻ 0.05 as compared with that from E. coli LPS treatment alone. Downloaded from that of the ultra pure R. sphaeroides LPS (supplemental Fig. S5B), ditions with 0.1% FCS in the presence or in the absence of 10– whereas the OKT3 mAb-induced activation of T cells did not in- 15% human serum. Using three different samples, Fig. 5C shows crease CRISPLD2 secretion although the proinflammatory medi- that human serum suppressed LPS-induced TNF-␣ release readily. ators, including TNF-␣ from PBMCs, were induced (supplemental However, the suppressive effect was reversed by the addition of Fig. S5A), suggesting that the activation of TLR4 on T cells can the Ab against CRISPLD2, but not the control Ab. The evidence

directly lead to the up-regulation of CRISPLD2 secretion. The revealed that the endogenous CRISPLD2 in sera down-regulated http://www.jimmunol.org/ collective results, as shown in Fig. 4 and supplemental Fig S5, A LPS-induced TNF-␣ production. To further confirm this result, and B, seem to exclude the possibility that the up-regulation of rCRISPLD2 was added into the culture system in the presence or CRISPLD2 secretion is a secondary response, such as proinflam- absence of 10% human serum. Fig. 5D demonstrates that the in- matory mediator-induced response, or a side effect of contamina- creased concentrations of rCRISPLD2 inhibit LPS-induced TNF-␣ tion, such as the direct stimulation of Ags via the TCR/CD3 com- production in a dose dependent manner and that the anti- plex or unknown molecules (Ͻ20 kDa). CRISPLD2 Ab reverses the inhibitory effects of not only endog- However, we cannot exclude the possibility that the LPS-in- enous CRISPLD2 but also those of additional rCRISPLD2 in cul- duced up-regulation of CRISPLD2 secretion can be mediated by ture medium. the activation of other LPS-receptors such as CD6, which binds to by guest on September 29, 2021 LPS and LTA, respectively, via TLR4-independent and nonover- CRISPDL2 protects mice against endotoxic shock lapping extracellular sites and then delivers intracellular signaling As the above experiments demonstrated that CRISPLD2 specifically in the presence of LPS (17). The question of whether LPS-acti- inhibits biological functions of LPS, we examined whether vated CD6 can up-regulate CRISPLD2 secretion should be further CRISPLD2 can ameliorate the devastating effects of systemic LPS investigated in the future. action in generating toxic shock in mice. Fig. 6A revealed that one i.p. injection of 450 ␮gofE. coli LPS killed 80% of the treated animals CRISPLD2 is a major biological regulator of LPS function within a few days. Injection of E. coli LPS in combination with 1.4 It is possible that endogenous CRISPLD2 may down-regulate LPS mg of rCRISPLD2 reduced the death rate to Ͻ20%, suggesting that immunostimulatory activities. If indeed the LPS sensitivity of CRISPLD2 exerts life-saving effects of anti-endotoxin in vivo (Fig. monocytes in PBMCs was to correlate negatively with the avail- 6A). By contrast, CRISP-3 as a control has no protective effect. ability of endogenous CRISPLD2 in culture medium, then exper- It has previously been shown that pretreatment of mice with small, imentally interfering with the interaction between LPS and nontoxic doses of LPS render recipients resistant to a lethal challenge CRISPLD2 should increase LPS sensitivity. with LPS (45, 46). This finding was consistent with our notion that To test this hypothesis, human PBMCs were incubated with LPS stimulates the generation of endogenous CRISPLD2. We there- increasing concentrations of LPS in the presence or absence of fore considered the possibility that CRISPLD2 elevation induced by a CRISPLD2-specific Abs, and then the release of CRISPLD2 and low dose of LPS may account for the protection against the lethal TNF-␣ were examined 18 h later. The experiment revealed that effect of a high dose of LPS. We found that the serum of mice that PBMCs spontaneously released CRISPLD2 into culture medium have been treated with a small dose of LPS more than doubled its and that the higher doses of LPS, Ͼ10 ng/ml, up-regulated the CRISPLD2 expression (Fig. 4G and Table III). At the same time, we release (Fig. 5A). In concordance with our hypothesis, the efficacy also noticed that CRISPLD2 was not the only LBP that accumulated of LPS was increased in the presence of Ab against CRISPLD2 as in the serum. LPS-binding IgG rose by an even higher margin (sup- compared with the control Ab, suggesting indeed that the anti- plemental Fig. S6). Table III showed that under such conditions mice CRISPLD2 Ab can remove an effective safety device against LPS experienced substantial protection against LPS-induced lethal shock. (Fig. 5B). Plotted against a lethal LPS dose, it was shown that CRISPLD2 serum Intruding germs reach their cellular targets via the bloodstream. levels as well as LPS-reactive IgG serum levels correlate similarly and Previous analysis revealed substantial but varying CRISPLD2 reciprocally with LPS lethality (Fig. 6B and supplemental Fig. S6). quantities in the serum of humans (Fig. 2A). It seemed of interest However, it has been known preadministration of the J5 LPS vaccine to determine whether CRISPLD2 molecules contained in the se- increases the concentration of serum anti-LPS core Abs in individuals rum provide animals and humans with a natural shield against the (20). Whether both LPS-binding structures synergistically account for effects of LPS. Human PBMCs were challenged under culture con- enhanced LPS resistance is to be further determined. 6654 SERUM LIPOPOLYSACCHARIDE-BINDING PROTEIN CRISPLD2 Downloaded from http://www.jimmunol.org/

FIGURE 5. Endogenous CRISPLD2 interferes with LPS-induced TNF-␣ response. A, PBMCs were stimulated with the increased concentration of E. coli LPS as indicated. The endogenous CRISPLD2 released in the culture supernatants was quantified at 18 h by ELISA, and the medium cultured without cells was used as a negative control. B, In the same experiment, anti-CRISPLD2 polyclonal Abs (Ⅲ) and unrelated polyclonal Abs (E) were added into cell cultures as compared with cultures without Abs (F). TNF-␣ released in the culture supernatants at 18 h was measured by ELISA. C, Human serum (HS) down-regulates LPS-induced TNF-␣ production. Sera from three healthy volunteers were used in the experiment. PBMCs were stimulated by E. coli LPS (100 ng/ml) with anti-CRISPLD2 polyclonal Ab or a control Ab (both 500 ␮g/ml) in the presence or absence of 15% human serum. TNF-␣ in culture supernatants were measured at 18 h by ELISA by guest on September 29, 2021 according to the protocol provided by the manufacturer (R&D Systems); LPS alone (open bars), LPS with control IgG (gray bars), LPS with the anti-CRISPLD2 p Ͻ 0.005 vs absence of human serum. D, Anti-CRISPLD2 polyclonal Abs against endogenous ,ءءء ;p Ͻ 0.05 ,ءء ;polyclonal Ab (black bars) are shown CRISPLD2 and rCRISPLD2 unleashes LPS immunostimulatory activities. In the same culture system with or without human serum (HS) 10% (#11), PBMCs were stimulated by E. coli LPS (10 ng/ml) in the presence of anti-CRISPLD2 polyclonal Ab or a control Ab (both 500 ␮g/ml) with additional rCRISPLD2 at the indicated concentrations. TNF-␣ released in the culture supernatants at 18 h was measured by ELISA. LPS with human serum (open bars), LPS with human serum ;p ϭ 0.048 ,ءء ;plus control IgG (gray bars), LPS with human serum plus anti-CRISPLD2 Ab (black bars), and LPS with only 0.1% FCS (diagonal bars) are shown .(p ϭ 0.00036; vs presence of anti-CRISPLD2 Ab (mean of three experiments Ϯ SD ,ءءء and

Discussion that CD6 acts as negative regulator of LPS function and endotoxic CRISPLD2 is a molecule recognized in the literature, but no LPS- shock induction (17). However, it may be argued, in favor of related functions are attributed to it (21–25). We were intrigued by CRISPLD2, that CD6 is highly expressed on cell membranes and the fact that it contains two LCCL domains, which are character- poorly present in serum (16), whereas CRISPLD2 lacking mem- istic LPS-binding structures (26). Our data indeed show, for the brane expression is amply represented in the serum. The downside first time, that CRISPLD2 is a potent LPS-binding protein that of LPS-reactive Abs is the fact that they are primarily directed exhibits significant LPS-binding affinity. against the immunodominant species-specific oligosaccharide side A major LPS-binding protein in the mammalian body, one chains, in contrast to CRISPLD2, and protect selectively, namely known to guide the transduction of signals involved in eliciting against the immunizing strain. The immunological effects of LPS LPS-dependent immune functions, is LBP (6, 7). CRISPLD2 are associated with the core glycolipid region in which there is shares with LBP none of the tasks examined here. On the contrary, little strain variation. Experimental vaccines have been produced the postulate that it could oppose LBP-mediated immunostimula- using an E. coli J5 mutant that lacks side strains to its core. J5- tion is supported by the finding that the polyclonal Ab against the specific Abs have been demonstrated to provide some protection CRISPLD2 region containing LCCL domains unleashes LPS im- against endotoxic shock (20). munostimulatory activities (Fig. 5). The mechanism of this antag- Given the fact that CRISPLD2 effectively blocks LPS-induced onism is unclear. CRISPLD2 could inhibit LBP-mediated immune immune functions, the uneven organ distribution of the molecule functions by competing for the target molecule, by facilitating neg- in the body may indicate that organs expressing high levels of ative signaling, or by using both mechanisms. CRISPLD2 would benefit from a persistent down-modulation of CRISPLD2 is not the only molecule the body may engage in the LPS immunostimulatory activity. The desirability of an abundant fight against bacterial LPS. We noted in our experiments that LPS- presence of CRISPLD2 in the placenta would seem evident from reactive Igs have a similar effect. Recently, it was also reported the fact that LPS is a strong inducer of fetal abortion (47). The Journal of Immunology 6655

Table III. Correlation between serum Crispld2 level and LPS sensitivity

LPS LPS Pretreatment Serum Crispld2 Administration Outcome (0.03 mg) (OD 450–570 nm)a (mg/mouse)b (Survival/total mice)c

No 0.134–0.166 0.5 0/4 No 0.130–0.201 0.8 0/4 Yes 0.223–0.327 0.5 3/4 Yes 0.323–0.446 0.8 4/6 Yes 0.338–0.386 1.1 4/4 Yes 0.351–0.371 1.4 0/2

a Serum Crispld2 was detected by ELISA assays on the 8th day after LPS pretreatment. b LPS was injected i.p. on the 10th day after LPS pretreatment. c Outcome on the 15th day after LPS pretreatment.

cess of a systemic functional presence of LPS in Gram-negative bacteria. The prevention of endotoxin shock by CRISPLD2 is un- equivocally demonstrated by our findings in the experimental

mouse model. Downloaded from A drawback in conventional sepsis treatment is the fact that, once the first indications of an immanent shock are detected, the avalanche of immunological events is already in full motion and it can no longer be stopped. The most effective protection against these avalanches lies in preventing their initiation. If we were to

draw the conclusion from our experiments that the risk of acquir- http://www.jimmunol.org/ ing Gram-negative sepsis is high when CRISPLD2 serum levels are low and, vice versa, the risk is low when CRISPLD2 serum levels are high, it would logically follow that the best precaution a FIGURE 6. rCRISPLD2 protects mice against endotoxin shock. A, physician can take against the onset of Gram-negative sepsis is to Mice were injected i.p. with E. coli LPS (450 ␮g/mouse, 22.5 mg/kg; n ϭ 22) alone or in combination with rCRISPLD2 (1.4 mg/mouse, 70 mg/kg; assure that a patient possesses sufficient amounts of CRISPLD2 in n ϭ 17) or CRISP-3 (0.64 mg/mouse, 32 mg/kg; n ϭ 7). The percentages his or her serum. We noted in these mice (Table III) that small of surviving animals are plotted in a Kaplan-Meyer survival curve; p Ͻ nontoxic doses of LPS boost the serum expression of CRISPLD2 0.001 vs LPS with PBS or CRISP-3. B, The lethal LPS doses correlate and protect animals against the lethal effect of the immunostimu- positively with serum Crispld2 concentrations. Serum CRISPLD2 was latory agent. We recognize that E. coli LPS itself can hardly be by guest on September 29, 2021 measured by ELISA on the 8th day after i.p. preinjection with or without considered a desirable therapeutic reagent, but it is conceivable a nontoxic dose of LPS (0.03 mg/mouse). Lethal doses of LPS (0.5, 0.8, that other less toxic microbial products or preparations may also 1.1, or 1.4 mg/mouse) were injected i.p. on the 10th day after i.p. prein- up-regulate CRISPLD2 serum levels. A clinical concept of guard- jection. The outcome was accounted on the 15th day after LPS pretreat- ing against Gram-negative sepsis would thus entail the careful ment. Serum Crispld2 in surviving mice was examined again on the 30th tracking of CRISPLD2 serum levels in patients with increased risk day, surviving mice received a third injection of LPS (0.9, 1.2 or 1.5 mg/ mouse) on the 32nd day, and the outcome was accounted again on the 37th of developing Gram-negative septic shock and attempts to boost day. The data of lethal LPS dose vs serum Crispld2 concentration from CRISPLD2 expression once its level falls below a critical each mouse were recorded. Together, all data were plotted by lethal LPS threshold. doses vs serum Crispld2 concentrations. Value of p ϭ 0.00009 and value of F ϭ 35.4. GraphPad Prism 5 software was used for statistic analysis. In Acknowledgments regression analysis, logarithmic trendline program in Excel was best-fit to We thank Q. N. Zhuang (GE Healthcare) for technical assistance on SPR. the values array in the plot, with the correlation coefficient R2 indicated on the graph. Disclosures The authors have no financial conflict of interest.

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