Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: FIGURE 1. CRISPLD2 binds LPS but not S. aureus LTA. A, Reaction with S. enterica serovar Minnesota (S. minnesota) and E. coli LPS shifts the mobility of rCRISPLD2 in a native PAGE mobility shift assay. B and C, Sensorgrams of E. coli LPS (B) and S. aureus LTA (C) binding to immobilized CRISPLD2. Either E. coli LPS or S. aureus LTA (0.3, 1.0, 3.0, 10, and 30 M) was passed over immobilized CRISPLD2. The dis- sociation constant of LPS, KD, was calculated as the ratio of these two constants (koff/kon) according to the binding parameters from a sensor- gram. D, RU that reflects E. coli LPS binding activity was measured by a BIAcore instrument when E. coli LPS (1.0, 3.0, 10, 30, and 100 M) was passed over the CRISPLD2 chip. E, Scatchard plot analysis and the KD of E. coli LPS binding to CRISPLD2 (mean SD).

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Clear Native PAGE, Mobility Shift, Binding Assay, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: FIGURE 2. CRISPLD2 occurs in cells and in serum. A, Immunoblots (IB) of 1/10 diluted sera from healthy volunteers (right; n 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 3) with the levels infant umbilical cord blood (n 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 the loading control. RNA extracted from CHO cell line with stable CRISPLD2 transfection was used as positive control (), and clean water was used as a negative control (). 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.

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Western Blot, Comparison, Clinical Proteomics, Imaging, Recombinant, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Positive Control, Negative Control, Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: 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- 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 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- 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 () was used as a positive control.

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Binding Assay, Labeling, Negative Control, Incubation, Cytometry, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: 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- ulated with or without E. coli LPS, and the CRISPLD2 concentrations in culture medium were quantitated by ELISA (mean of three experiments SD). F and G, Up-regulation of Crispld2 expression in mouse serum after administration of a nontoxic 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 PBS for PAGE separation, so real concentrations of Crispld2 are 10 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.

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: 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 () 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 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 polyclonal Ab (black bars) are shown; , p 0.05; , p 0.005 vs absence of human serum. D, Anti-CRISPLD2 polyclonal Abs against endogenous 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 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 0.048; and , p 0.00036; vs presence of anti-CRISPLD2 Ab (mean of three experiments SD).

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The novel lipopolysaccharide-binding protein CRISPLD2 is a critical serum protein to regulate endotoxin function.

doi: 10.4049/jimmunol.0802348

Figure Lengend Snippet: FIGURE 6. rCRISPLD2 protects mice against endotoxin shock. A, Mice were injected i.p. with E. coli LPS (450 g/mouse, 22.5 mg/kg; n 22) alone or in combination with rCRISPLD2 (1.4 mg/mouse, 70 mg/kg; n 17) or CRISP-3 (0.64 mg/mouse, 32 mg/kg; n 7). The percentages of surviving animals are plotted in a Kaplan-Meyer survival curve; p 0.001 vs LPS with PBS or CRISP-3. B, The lethal LPS doses correlate positively with serum Crispld2 concentrations. Serum CRISPLD2 was measured by ELISA on the 8th day after i.p. preinjection with or without a nontoxic dose of LPS (0.03 mg/mouse). Lethal doses of LPS (0.5, 0.8, 1.1, or 1.4 mg/mouse) were injected i.p. on the 10th day after i.p. prein- jection. The outcome was accounted on the 15th day after LPS pretreat- ment. Serum Crispld2 in surviving mice was examined again on the 30th 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 day. The data of lethal LPS dose vs serum Crispld2 concentration from each mouse were recorded. Together, all data were plotted by lethal LPS doses vs serum Crispld2 concentrations. Value of p 0.00009 and value of F 35.4. GraphPad Prism 5 software was used for statistic analysis. In regression analysis, logarithmic trendline program in Excel was best-fit to the values array in the plot, with the correlation coefficient R2 indicated on the graph.

Article Snippet: The membranes were blocked with 5% dry skim milk and incubated with rabbit anti-CRISPLD2 polyclonal Ab at 37°C for 2 h, followed by a secondary Ab coupled with fluorescein (Rockland) at 37°C for 1 h. The blotting membranes were finally scanned by the Odyssey infrared imaging system (LICOR Biosciences).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Software