Identification of Evasin-3 and -4. (a) Identification of Evasin-3 and -4 by cross-linking to radiolabeled chemokine in supernatants from transfected HEK293 cells. Viral CHBP p35 incubated with [¹²⁵I]-CCL2 as a positive control in the presence (lane 1) or absence (lane 2) of the cross-linker BS₃; supernatant from clone 69.19.1 incubated with [¹²⁵I]-CXCL8 and BS₃ (lane 3); supernatant from clone 10.27.1 incubated with [¹²⁵I]-CCL5 and BS₃ (lane 4) or [¹²⁵I]-CCL11 and BS₃ (lane 5). (b) Alignment of the predicted protein sequences of Evasin-1 and -4. Conserved residues are highlighted in gray and cysteines are shown in bold type. The predicted protein sequence of Evasin-3 is shown below the alignment. The signal peptides are shown in italics. Possible signal peptides of Evasin-4 are shown as follows: based on SignalJ prediction, black underline; based on secondary structure prediction, dotted underline; based on similarity to Evasin-1, dashed line. (c) Determination of the selectivity of immobilized recombinant Evasin-3 produced in E. coli by surface plasmon resonance. Sensograms corresponding to CXCL8 (bold black lines) CXCL1 (dotted black lines), MIP-2 (gray lines), and KC (dotted gray lines) showed strong binding of these chemokines to Evasin-3. The sensograms corresponding to CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL18, CCL27, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, and CX₃CL1 (black lines) indicated no binding of these chemokines to Evasin-3.

Evasin-1 inhibits cell recruitment in vitro and in vivo. (a) Inhibition of CCL3- (▪), CCL3L1- (•) and CCL4-mediated (▴) chemotaxis of L1.2/CCR5 transfectants. The IC₅₀ values are 2 nM for CCL3, 0.17 nM for CCL3L1, and 4 nM for CCL4. Data are shown as the mean ± the SD as a percentage of maximal recruitment and are representative of three to five individual experiments. (b) Evasin-1 inhibits granulocyte recruitment induced by CCL3 in mice. Evasin-1 was administered s.c. 45 min before the injection of 10 μg of CCL3 i.p. Cells in the peritoneal cavity were counted 18 h after the injection of CCL3. One representative experiment of three is shown, with 8 mice per group. **, P < 0.01 when compared with PBS-treated group. Error bars represent the mean ± SEM.

Evasin-1 inhibits cellular recruitment in an animal model of psoriasis. (a) Dermal and epidermal thickness of the skin of WT and D6-deficient (KO) mice after a single cutaneous application of TPA. Evasin-1 (30 μg/mouse) and anti-TNF-α were given daily, and tissues were collected on day 4 for histological assessment. One representative experiment of three is shown, with eight mice per group. ***, P < 0.001 when compared with PBS-treated group. Error bars represent the mean ± SEM. (b) Haematoxylin and eosin staining of sections from untreated skin of sham and TPA-injected D6-deficient mice treated with PBS, Evasin-1, or anti-TNF-α. Skin sections were obtained on day 4 after TPA application and are representative of 10 mice per group per experiment. Bars, 100 μm.

Evasin-3 inhibits cell recruitment in vitro and in vivo. (a) Inhibition of ¹²⁵I-CXCL8 binding to CXCR2 by Evasin-3 produced in HEK293 cells (•) and by Evasin-3 produced in E. coli (▪). (b) Inhibition of CXCL8 induced neutrophil chemotaxis in vitro. (c) Inhibition of KC induced neutrophil recruitment into the peritoneal cavity (c) and into the knee joint (d). There were six mice per group in each experiment. **, P < 0.01; ***, P < 0.001 when compared with PBS-treated group. Error bars represent the mean ± SEM.

Direct comparison of Evasin-3 and -1. (a) Inhibition of chemokine-induced neutrophil recruitment into the knee joint induced by chemokine (a) or by Zymozan (b). Evasin-1 (10 μg) and Evasin-3 (1 μg) were administered 30 min into the knee joint before the administration of 300 ng mMIP-1α or 30 ng KC, respectively, or 30 μg Zymosan. Cells in the joint were counted 6 h after the injection of stimuli. There were five mice per group in each experiment. **, P < 0.01; ***, P < 0.001 when compared with PBS-treated group. (c) Prevention of ischemic reperfusion. Evasin-1 and -3 were administered s.c. at the doses indicated 30 min before the reperfusion of the occluded superior mesenteric artery. There were seven to eight mice in each experimental group. **, P < 0.01 when compared with PBS-treated group. Error bars represent the mean ± SEM.

Chemokine binding activity in tick saliva. (a) Tick saliva inhibits the binding of CCL3, CCL5, and CXCL8 to their cognate receptors. Radiolabeled chemokines were incubated with CHO cell membranes expressing CCR1 for CCL3 and CCL5, and CXCR2 for CXCL8, in the presence of 1 μl, 10 μl, or in the absence of tick saliva. The antichemokine activity is caused by the presence of more than one binding protein because an excess of CXCL8 did not abolish the inhibition of CCL3 binding. Error bars represent the mean ± SEM. (b) Detection of CHPBs in tick saliva by SPR. Sensograms demonstrated binding to CCL5(E66A) and CCL3 immobilized on a CM4 chip. (c) Detection of distinct CHPBs to CCL5 and CXCL8 by Retentate chromatography SELDI of flight mass spectrometry (RC-SELDI-TOF-MS). Tick saliva was incubated on a PS-20 Proteinchip array coated with either CCL5 (bottom), CXCL8 (middle), or the control protein equine myoglobin (top). Specific peaks, indicated by arrows, corresponding to a protein with a mass of 11,052 Daltons binding to CCL5 and of 7,133 Daltons for the binding to CXCL8 were observed.

Crystal structures of Evasin-1 and -3. Ribbon diagram of Evasin-1 (a) and Evasin-3 (b) with the amino acid sequence depicting residues in loops (green), α-helix (red), or β-sheet (blue).

Evasin-1 inhibits cellular recruitment in models of Th1 and Th2 inflammation. (a) Evasin-1 inhibits granulocyte recruitment induced by antigen challenge of immunized mice in models of Th1-predominant delayed-type hypersensitivity and Th2-predominant late-phase response. In the Th1 model, 10 μg Evasin-1 was administered s.c. 45 min before intrapleural injection of 10 μg of mBSA in mice immunized with mBSA in Freund's complete adjuvant. Cells in the pleural cavity were counted 48 h after the injection of antigen. In the Th2 model, 10 μg Evasin-1 was administered s.c. 45 min before and 24 h after the intratracheal injection of 10 μg of S. mansoni egg antigen in mice immunized with S. mansoni eggs. Cells in the bronchoalveolar lavage fluid were counted 24 and 48 h after the injection of antigen. There were six mice per group in each experiment. *, P < 0.05; **, P < 0.01 when compared with PBS-treated group. (b) Evasin-1 blocks adhesion and emigration of leukocytes induced by mMIP-1α as assessed by intravital microscopy performed in the cremaster vein of C57BL/6 mice. Evasin-1 was injected s.c. in the right scrotal skin 1 h before exteriorization. Normalized data were analyzed by one-way analysis of variance, and differences between groups were assessed using Student-Newman-Keuls post-test. *, P < 0.05; **, P < 0.01. There were six mice per group in each experiment. Error bars represent the mean ± SEM.

Evasin-1 inhibits lethality, cellular recruitment, and fibrosis in a model of bleomycin-induced pulmonary injury. (a) Evasin-1 inhibits lethality associated with pulmonary instillation of bleomycin in C57BL/6 mice. Bleomycin was instilled intratracheally at doses of 0.0625 U (circles) or 0.125 U (squares) and survival rates were evaluated for 25 d. PBS (open symbols) or Evasin-1 (10 μg per animal; closed symbols) were administered s.c. 45 min before and every 12 h after initial bleomycin instillation. There were 12 mice per group in each experiment. *, P < 0.05 when comparing PBS- and Evasin-1–treated animals at 0.125 U. (b) Evasin-1 inhibits neutrophil recruitment induced by bleomycin. Neutrophil recruitment was evaluated at 2 and 8 d after bleomycin (0.125 U) and Evasin-1 (10 μg per animal) was administered s.c. 45 min before and every 12 h after initial bleomycin instillation. There were six mice per group in each experiment. *, P < 0.05 when compared with the PBS-treated group. Error bars represent the mean ± SEM. (c) Evasin-1 inhibits pulmonary inflammation and fibrosis induced by instillation of bleomycin. Hematoxylin and eosin– (top) and Gomori's trichrome–stained (bottom) sections from animals instilled with sodium chloride (0.9% NaCl, 25 μl) or bleomycin (0.125 U). Evasin-1 (10 μg per animal) was administered as previously described and lungs were removed on day 16. Bars: (top) 100 μm; (bottom) 50 μm.

Evasin-3 inhibits neutrophil recruitment and ameliorates inflammation and functionality in a mouse model of antigen-induced arthritis. (a–g) Reduction of inflammation in mBSA induced arthritis by Evasin-3 (1 μg per animal). (a) Inhibition of total cell accumulation in the synovial cavity. (b) Inhibition of neutrophil infiltration into the knee joint. (c) Reduction of hypernociception induced by intraarticular antigen challenge of immunized mice. (d) Reduction of TNF-α production in periarticular tissues. (e) Inhibition of adhesion of leukocytes to synovial microvessels as assessed by intravital microscopy. (f and g) Histological evaluation of the tissue sections form the knee joint. All parameters were evaluated 24 h after antigen challenge, and there were 6 mice per group in each experiment. *, P < 0.05; **, P < 0.01. Error bars represent the mean ± SEM. Bars, 100 μm.