Presence of a CKBP in S. mansoni ES. (A) Cercarial homogenates (1) or homogenates (2, 4, and 6) or secretions (3, 5, and 7) from schistosomula (2 and 3), worms (4 and 5), or eggs (6 and 7) or binding media (8) were cross-linked to ¹²⁵I-CXCL8 or ¹²⁵I-CCL3. All schistosome antigen preparations had nonspecific binding (*) to iodinated chemokines (55–60 kD) that was also detectable in the binding media. (B) CXCL8 and CCL3 binding to U937 cells in the presence of a range of protein concentrations of homogenates (open circles and dotted line) or secretions (closed circles and continuous line) from different life cycle stages of S. mansoni. (C) Cross-linking of ¹²⁵I-CXCL8 or ¹²⁵I-CCL3 to binding media (1 and 4) or smCKBP in the absence (2 and 5) or presence (3 and 6) of excess unlabeled chemokine. (D) Anti-smCKBP rabbit sera probe of Western blot of homogenates of S. mansoni cercariae (1), schistosomula (2), worms (3), and eggs (4), S. japonicum eggs (5), S. haematobium eggs (6), or secretions from S. mansoni schistosomula (7), worms (8), or eggs (9).

r-smCKBP is a functional CKBP. (A) Silver-stained SDS-PAGE gel of natural or r-smCKBP. Western blot of SDS-PAGE resolved natural (line 1) and r-smCKBP (line 2) probed with anti-smCKBP antisera or NRS. (B) r-smCKBP was cross-linked to ¹²⁵I-chemokines with or without competing cold chemokines. (C) Binding of ¹²⁵I-CXCL8 and ¹²⁵I-CCL3 to U937 cells after preincubation with r-smCKBP (red filled circles and line) or with a control protein (CRD, open circles and continuous line). (D) Migration of human neutrophils in response to CXCL8 preincubated with r-smCKBP (red bars) or with control CRD (open bars). Migration was also elicited with 100 nM n-FMLP preincubated with 1,000 ng/ml of r-smCKBP or CRD. The values shown represent means ± SD from triplicate samples. (E) CCL5-induced activation (Ca²⁺ flux) of human PBMCs from three donors treated with r-smCKBP (red filled circles and line) or control CRD (open circles and continuous line). Data are representative of two to four separate experiments.

r-smCKBP inhibits inflammation in vivo. (A) In an air pouch model, r-smCKBP treatment significantly inhibits CXCL8-induced neutrophil infiltration (P < 0.001). The percentages of neutrophils on cytospins are shown as means ± SEM (n = 4–6) and are representative of two separate experiments. (B) r-smCKBP inhibits inflammation in a contact hypersensitivity model. DNFB–sensitized mice were injected intravenously with r-smCKBP or CRD before application of DNFB solution. Data are presented as means ± SE of ear thickness (n = 6–8 mice). Significantly reduced inflammation was observed in mice treated with r-smCKBP 24 h after DNFB exposure (P < 0.001 by Student's t test). (C) Representative cross sections of ears and cytospins of the pinnae infiltrate (H&E stained). Data are representative of three separate experiments. (D) r-smCKBP blocks CXCL8-induced pulmonary neutrophilia. Mice were treated intranasally (i.n.) with CXCL8 or PBS or were injected intravenous (i.v.) with r-smCKBP or PBS. 18 h later, whole-body plethysmography was used to determine lung function (Penh) and bronchoalveolar lavage (BAL) fluid recovered for total cell counts and flow cytometry for neutrophil infiltration. Statistical values represent differences between CXCL8 + PBS–treated and CXCL8 + r-smCKBP–treated groups by Student's t test. All data represent means ± SE from four to seven mice per group. Data are representative of two separate experiments.

Secretion of smCKBP from live eggs in vitro and in vivo. (A) Precipitate formation (arrows) around live eggs cultured in vitro with anti-smCKBP sera but not NRS. (B) Detection of smCKBP (brown stain) within the granulomas surrounding eggs in liver or intestines of infected mice. Bar, 50 μm.

Blocking smCKBP in vitro increases the pulmonary granulomatous inflammation to live eggs. (A) Rabbit anti-smCKBP sera dose-dependently blocks r-smCKBP activity in vitro. Different dilutions, in triplicate, of NRS (open circles and continuous line) and anti-smCKBP sera (red filled circles and line) were incubated with r-smCKBP before addition of ¹²⁵I-CXCL8. Samples were then tested in a U937 cell binding assay. Data are presented relative to 100% blocking of ¹²⁵I-CXCL8 cell binding by r-smCKBP. (B) Sections of lungs showing granuloma around eggs (arrow) in lungs of mice treated with NRS or anti-smCKBP sera as stained by H&E. Bar, 50 μm. (C) Size of egg granuloma in lungs of mice treated with NRS or anti-smCKBP sera. The values shown represent means ± SD. (D) High magnification of cells within granuloma around eggs (top right, arrows) in lungs of mice treated with NRS or anti-smCKBP sera. Bar, 10 μm. (E) Cell composition of pulmonary granulomas around eggs in lungs of mice treated with NRS or anti-smCKBP sera. Data represent means ± SD from six mice per group and are representative of two separate experiments. Student's t test was used to test for statistical differences between groups, and p-values are shown.