T., Sette A., et al. the first demonstration of the major role played by this chemokine in quick neutrophil mobilization after contamination. Pre-activated OVA multimers FS reactive with amine residues thus provide an efficient carrier for auto-vaccination against 9C90 kDa autologous proteins. infection. MATERIALS AND METHODS Reagents and mice All vaccinations were performed in C57BL/6 mice maintained under specific pathogen-free conditions at our animal facility (Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium) under the supervision of Dr. Guy Warnier. The experimental protocol and animal handling were approved by the ethical committee of the Faculty of Medicine, Universit Catholique de Louvain. The experimental procedures with infection in C57BL/6 mice. GCP-2 Ab4, NIMP-R14, a mAb known to induce complete neutrophil depletion , or control antibody (500 g) was injected i.p. 24 h prior to ear i.d. infection with 106 test as indicated. RESULTS AND DISCUSSION Production of OVA-glutaraldehyde carrier Glutaraldehyde cross-links proteins by reacting with nonprotonated NH2 residues and rapidly forms insoluble polymers when incubation is performed at neutral or alkaline pH . We observed that OVA, incubated overnight with a 100-fold molar excess of glutaraldehyde at pH 6, formed large polymers, which in spite of their size, remained soluble. When fractionated by size exclusion chromatography, these conjugates (hereafter designated OVAglu) eluted in a broad peak 1000 kD in size (Fig. 1A). As glutaraldehyde is a multifunctional compound, we anticipated that OVA polymerized with an excess of cross-linking agent would be covered with free aldehyde groups that could react with free amines on other proteins. To favor this possibility, the reaction with a target protein was performed in 0.1 Allopurinol sodium M carbonate buffer at pH 8.5 and incubated for at least 6 h. The remaining free glutaraldehyde sites were subsequently saturated with a tenfold molar excess of PADRE peptide to provide additional T cell help. As Allopurinol sodium shown in Table 1, incubation of GCP-2 or GM-CSF with OVAglu at a 1:1 molar ratio resulted in the formation of conjugates that could be detected by ELISA using a combination of anti-OVA and anticytokine antibodies. Similar results were obtained for all complexes mentioned in this manuscript when specific antibodies against the target cytokine were available. These results prove that OVAglu multimers are highly reactive and stable. The major advantage of these polymers is that they minimize exposure of the target factor to the cross-linking agent as compared with reactions performed by mixing OVA, cytokine, and free cross-linker. Open in a separate window Figure 1. OVAglu polymers, anti-GCP-2COVAglu auto-vaccination, and role of GCP-2 in neutrophil mobilization in infection.(A) OVAglu polymers fractionated by size exclusion chromatography. Size markers are mouse IgG1 (150 kD) and OVA (45 kD). (B) Detection by ELISA of anti-GCP-2 antibodies in sera from C57BL/6 mice vaccinated with GCP-2COVAglu. (C) GCP-2 ELISA performed with two mAb derived from auto-vaccinated mice: GCP-2 Ab1 for coating and biotinylated GCP-2 Ab2 for detection. (D) Inhibition by GCP-2 Ab4 (0.5 mg/ml) of Ca2+ mobilization induced by GCP-2 (60 ng/ml) in human neutrophils. Ca2+ fluxes from one representative experiment out of three are shown. (E) Impairment of neutrophil mobilized 24 h after infection with in the ear dermis of C57BL/6 mice treated with GCP-2 Ab4, neutrophil-depleting antibody NIMP-R14, or IgG2b control (CTR) antibody (meansem of six/group). These results Allopurinol sodium are representative of three independent experiments. *test). Table 1. Detection of CytokineCOVA Complexes parasites. The number of neutrophils migrating out of infected ear dermis 24 h after infection was inhibited (65%; infection are still not well defined and may involve several chemokines and cytokines such as MIP-2(CXCL2), keratinocyte-derived chemokine (CXCL1), and IL-17 . The inhibition of neutrophil recruitment by anti-GCP-2 Ab4 reported here is, to our knowledge, the first demonstration of the role played by this chemokine in the early neutrophil mobilization in this infection. Anticytokine auto-vaccination To further evaluate the efficacy of OVAglu in breaking tolerance against self-antigens, attempts were made to vaccinate mice with OVAglu conjugated to various cytokines: GM-CSF, IL-17F, IL-17E/IL-25, IL-27p28, EBI3, and TGF-1. In all cases, specific antibodies.