Patent Application: US-8727302-A

Abstract:
the present invention provides novel nucleic acids , novel polypeptide sequences encoded by these nucleic acids , methods for production thereof , and uses thereof , for a novel elr - cxc chemokine receptor antagonist .

Description:
( the following abbreviations are used throughout this disclosure : ards , acute respiratory distress syndrome ; balf , bronchoalveolar lavage fluid ( s ); bhr , bolton - hunter reagent ; cxcr1 , cxcr2 , cxcl8 receptors a , b , respectively ; elr , glutamic acid - lysine - arginine motif ; cxcl1 , growth - related oncogenealpha ; cxcl4 , platelet factor - 4 ; cxcl5 , epithelial - derived neutrophil activator - 78 ; cxcl6 , granulocyte chemotactic protein - 2 ; cxcl8 , interleukin - 8 ; fmlp , formyl methionyl - leucylproline bacterial tripeptide ; iptg , isopropyl - thio - d - galactopyranoside ; mip - 2 , macrophage inflammatory protein - 2 ; pmsf , phenylmethylsulfonyl fluoride ; tmb , tetramethylbenzidine .) when amino terminal truncation of bovine cxcl8 is combined with a lysine to arginine substitution at amino acid 11 ( i . e ., cxcl8 ( 3 - 74 ) k11r ), dramatic increases in cxcr1 and cxcr2 receptor affinity are evident , such that cxcl8 ( 3 - 74 ) k11r competitively inhibits the binding of multiple ligands to both receptors ( ref . 24 ). further truncation into the receptor - signaling elr motif ( e . g ., amino acids 4 – 6 of human cxcl8 ) of some cxc chemokines can transform them into mild ( cxcl8 ( 6 - 72 ) ) to moderate ( cxcl1 ( 8 - 73 ) ) receptor antagonists ( ref . 15 , 25 ). as disclosed herein , the introduction into bovine cxcl8 ( 3 - 74 ) k11r of a second amino acid substitution , glycine 31 to a proline residue ( i . e ., cxcl8 ( 3 - 74 ) k11r / g31p ), renders this cxcl8 analogue a very high affinity antagonist of bovine and human elr - cxc chemokine responses . it fully antagonizes the entire array of elr - cxc chemokines expressed within bacterial or endotoxin - induced inflammatory foci and blocks endotoxin - induced inflammation in vivo . although the following discussion deals primarily with bovine neutrophils , other mammalian ( including human ) inflammatory cells also display cxcr1 and cxcr2 receptors ( see , for example , ref . 52 ) and so are vulnerable to inhibition by cxcl8 ( 3 - 74 ) k11r / g31p . accordingly , the present invention has broad applicability to mammalian elr - cxc chemokine - mediated pathologies . in an alternate embodiment of the invention , it is envisioned that compounds having the same three dimensional structure at the binding site may be used as antagonists . three dimensional analysis of chemical structure is used to determine the structure of active sites , including binding sites for chemokines . chemical leads with high throughput screening have been used to generate and chemically optimize a selective antagonist of the cxcr2 ( ref . 17 ). a similar approach was also used to generate a ccr3 antagonist ( ref . 56 ). wells et al ( ref . 57 ), has employed nuclear magnetic resonance spectroscopy ( nmr ) to detail the three dimensional structure of ligands for cxcr , including both elr and non - elr cxc chemokines . with their nmr information , wells et al generated multiple substitutions within the receptor binding sites of multiple chemokines , such that they could substantially alter the ligands &# 39 ; receptor specificities . reagents & amp ; supplies . the following reagents were purchased commercially : glutathione - sepharose , the expression vector pgex - 2t , sephadex g - 25 ( amersham - pharmacia - biotech , baie d &# 39 ; urfé , pq ), bolton - hunter reagent , a protein biotinylation kit ( pierce scientific , rockford , ill . ), the sequencing vector pbluescript ii ks , pfu turbo ™ dna polymerase ( stratagene , la jolla , calif . ), a site - directed mutagenesis kit ( quickchange ™; boerhinger - mannheim canada , laval , pq ), aprotinin , benzene , calcium ionophore a23187 , chloramine t , cytochalasin b , dimethylformamide , endotoxin ( escherichia coli lipopolysaccharide , serotype 0127b8 ), isopropyl - thio - d - galactopyranoside ( iptg ), leupeptin , p - nitrophenyl - β - d - glucuronide , mineral oil , silicon oil , tetramethylbenzidine ( tmb ), phenylmethylsulfonyl fluoride ( pmsf ), phorbol - 12 , 13 - myristate acetate ( pma ), and triton x - 100 ( sigma chemical co , mississauga , on ), a diff - quick staining kit ( american scientific products , mcgaw pk , ill . ), human cxcl1 , cxcl5 , and cxcl8 ( r & amp ; d systems inc , minneapolis , minn . ), horse radish peroxidase ( hrp )- conjugated anti - rabbit ig ( zymed , south san francisco , calif . ), dmem , hbss ( gibco , grand island , n . y . ), hrp - streptavidin ( vector labs , burlingame , calif . ), abts enzyme substrate ( kirkegaard & amp ; perry labs , gaithersburg , md . ), bovine serum albumin ( bsa ), and lymphocyte separation medium ( icn pharmaceuticals , aurora , ill .). generation of cxcl8 . sub . ( 3 - 74 ) k11r analogues . the high affinity cxcr1 / cxcr2 ligand cxcl8 . sub . ( 3 - 74 ) k11r , and its t12s / h13f analogue were generated in accordance with the methods described in li and gordon ( ref . 24 ). the gly31pro ( g31p ), pro32gly ( p32g ), and g31p / p32g analogues of these proteins were similarly generated by site - directed mutagenesis using pcr with the appropriate forward and reverse oligonucleotide primers ( table 1 ). the products from each reaction were digested with dpni , ligated into the vector pgex - 2t , transfected into hb101 cells , and their sequences verified commercially ( plant biotechnology institute , saskatoon ). briefly , the recombinant bacteria were lysed in the presence of a protease inhibitor cocktail ( 2 mm pmsf , 2 μg / ml aprotinin , and 2 μg / ml leupeptin ) and the recombinant fusion proteins in the supernatants purified by affinity chromatography , using glutathione - sepharose beads in accordance with the methods of caswell et al . ( ref . 26 ). the cxcl8 ( 3 - 74 ) k11r analogues were cleaved from the gst fusion proteins by thrombin digestion , dialysed against phosphate buffered saline ( pbs ), run through commercial endotoxin - removal columns , and then characterized by polyacrylamide gel electrophoresis ( page ) and western blotting with a goat anti - bovine cxcl8 antibody ( provided by dr . m . morsey ). each purified analogue had a molecular mass of 8 kda , was specifically recognized by the anti - cxcl8 antibody in the western blotting , and had a relative purity of 96 %, as determined by densitometric analysis of the page gels . labeling of the recombinant proteins . we used biot cxcl8 for the initial surveys of analogue binding to neutrophils and 125 i - cxcl8 for the later stage assays of relative receptor affinity . cxcl8 was biotinylated and the levels of biotin substitution determined using a commercial kit , as noted in li and gordon ( ref . 24 ). the biot cxcl8 was substituted with 2 . 15 moles of biotin per mole of cxcl8 . cxcl8 was radiolabeled with 125 i using the bolton - hunter reagent ( bhr ) method , as noted in detail ( ref . 24 ). the labeled protein was separated from the unincorporated 125 i - bhr by chromatography on sephadex g50 , and the labeled cxcl8 characterized for its relative affinity for neutrophils and the time required to achieve binding equilibrium , as noted in li and gordon ( ref . 24 ). cxcl8 ( 3 - 74 ) k11r analogue binding assays . cells ( 85 – 93 % neutrophils ) were purified from the blood of cattle in accordance with the caswell method ( ref . 26 ). in preliminary experiments , we determined that none of our analogues affected the viability of neutrophils , as determined by trypan blue dye exclusion . for the broad analogue surveys , neutrophils in hbss / 0 . 5 % bsa were incubated for 2 h at 4 ° c . with the analogue , washed in cold dmem , and then incubated for another 2 h at 4 ° c . with biot cxcl8 ( 1000 ng / ml ). the cell - associated biotin was detected by incubating the washed cells with alkaline phosphatase - conjugated streptavidin ( 1 : 700 dilution ) and then with abts enzyme substrate . the od 405 of the samples was determined using an elisa plate reader . medium - treated neutrophils routinely bound sufficient . sup . biotcxcl8 to generate an od 405 of 0 . 5 – 0 . 6 . for the in - depth studies with cxcl8 ( 3 - 74 ) k11r / g31p , we used 125 i - cxcl8 in binding inhibition assays with unlabeled cxcl8 or cxcl8 ( 3 - 74 ) k11r / g31p . in preliminary experiments we determined that the binding equilibrium time of neutrophils for 125 i - cxcl8 was 45 min and that 20 pm 125 i - cxcl8 just saturated the cell &# 39 ; s high affinity receptors . thus , in our assays , 10 6 purified neutrophils were incubated for 45 min on ice with 20 pm 125 i - cxcl8 and varying concentrations of unlabeled competitor ligand . the cells were then sedimented through 6 % mineral oil in silicon oil and the levels of cell - associated radio - ligand determined using a counter . the non - specific binding of 125 icxcl8 to the cells was assessed in each assay by including a 200 - fold molar excess of unlabeled ligand in a set of samples . this value was used to calculate the percent specific binding ( ref . 27 ). neutrophil β - glucuronidase release assay . the neutrophil β - glucuronidase assay has been reported in detail ( ref . 24 ). briefly , cytochalasin b - treated neutrophils were incubated for 30 min with the cxcl8 analogues , then their secretion products assayed colorimetrically for the enzyme . β - glucuronidase release was expressed as the percent of the total cellular content , determined by lysing medium - treated cells with 0 . 2 % ( v / v ) triton x - 100 . neutrophil challenge with the positive control stimulus pma ( 50 ng / ml ) and a23187 ( 1 μg / ml ) induced 42 +/− 6 % release of the total cellular β - glucuronidase stores . samples from inflammatory lesions . we obtained bronchoalveolar lavage fluids ( balf ) from the lungs of cattle ( n = 4 ) with diagnosed clinical fibrinopurulent pneumonic mannheimiosis ( ref . 8 ), as well as teat cistern wash fluids from cattle ( n = 4 ) with experimental endotoxin - induced mastitis ( ref . 28 ). in preliminary dose - response experiments we determined that 5 μg of endotoxin induced a strong ( 70 – 80 % maximal ) mammary neutrophil response . thus , in the reported experiments mastitis was induced by infusion of 5 μg of endotoxin or carrier medium alone ( saline ; 3 ml volumes ) into the teat cisterns of nonlactating holstein dairy cows , and 15 h later the infiltrates were recovered from the cisterns by lavage with 30 ml hbss . the cells from the balf and teat cistern wash fluids were sedimented by centrifugation and differential counts performed . untreated and cxcl8 - depleted ( below ) wash fluids were assessed for their chemokine content by elisa ( cxcl8 only ) and chemotaxis assays . neutrophil chemotaxis assays . microchemotaxis assays were run in duplicate modified boyden microchemotaxis chambers using polyvinylpyrrolidone - free 5 μm pore - size polycarbonate filters , in accordance with known methods ( ref . 26 , 29 ). for each sample , the numbers of cells that had migrated into the membranes over 20 – 30 min were enumerated by direct counting of at least nine 40 . times . objective fields , and the results expressed as the mean number of cells / 40 × field (+/− sem ). the chemoattractants included bovine or human cxcl8 , human cxcl5 and cxcl1 , pneumonic mannheimiosis balf and mastitis lavage fluids ( diluted 1 : 10 – 1 : 80 in hbss ), while the antagonists comprised mouse anti - ovine cxcl8 antibody 8m6 ( generously provided by dr . p . wood , csiro , australia ) or the cxcl8 ( 3 - 74 ) k11r analogues . in some assays we preincubated the samples with the antibodies ( 5 μg / ml ) for 60 min on ice ( ref . 30 ). in others we generated cxcl8 - specific immunoaffinity matrices with the 8m6 antibodies and protein - a - sepharose beads and used these in excess to absorb the samples ( ref . 8 , 31 ); the extent of cxcl8 depletion was confirmed by elisa of the treated samples . for assays with the recombinant antagonists , the inhibitors were mixed directly with the samples immediately prior to testing . cxcl8 elisa . for our elisa , mab 8m6 was used as the capture antibody , rabbit antiovine cxcl8 antiserum ( also from p . wood , csiro ) as the secondary antibody , and hrpconjugated anti - rabbit ig , and tmb as the detection system , as noted in caswell et al . ( ref . 8 ). serial dilutions of each sample were assayed in triplicate , and each assay included a recombinant bovine cxcl8 standard curve . cxcl8 ( 3 - 74 ) k11r / g31p blockade of endotoxin responses in vivo . we used a sequential series of 15 h skin tests to test the ability of cxcl8 ( 3 - 73 ) k11r / g31p to block endotoxininduced inflammatory responses in vivo . for each test , we challenged 2 week - old healthy holstein cows intradermally with 1 μg endotoxin in 100 μl saline , then 15 h later took 6 mm punch biopsies under local anaesthesia ( lidocaine ) and processed these for histopathology ( ref . 31 ). following the first ( internal positive control ) test , we injected each animal subcutaneously , intramuscularly , or intravenously with cxcl8 ( 3 - 74 ) k11r / g31p ( 75 μg / kg ) in saline , then challenged them again with endotoxin , as above . the animals were challenged a total of 4 times with endotoxin , such that 15 h reaction site biopsies were obtained at 0 , 16 , 48 , and 72 h post - treatment . the biopsies were processed by routine methods to 6 . mu . paraffin sections , stained with giemsa solution , and examined in a blinded fashion at 400 − magnification ( ref . 31 , 32 ). the mean numbers of neutrophils per 40 × objective microscope field were determined at three different depths within the skin , the papillary ( superficial ), intermediate , and reticular ( deep ) dermis . statistical analyses . multi - group data were analyzed by anova and post - hoc fisher protected least significant difference ( plsd ) testing , while two - group comparisons were made using the students t - test ( two - tailed ). the results are expressed as the mean +/− sem . cxcl8 ( 3 - 74 ) k11r / g31p competitively inhibits cxcl8 binding to neutrophils . we surveyed the ability of each cxcl8 ( 3 - 74 ) k11r analogue to bind to the cxcl8 receptors on neutrophils , and thereby compete with cxcl8 as a ligand . in our initial surveys , we employed biot cxcl8 binding inhibition assays , incubating the cells with the analogues ( 10 ng / ml ) for 2 h at 4 ° c . prior to exposure to biot cxcl8 ( 1 μg / ml ). this level of cxcl8 approximates those found in the lung tissues of sheep with experimental pneumonic mannheimiosis ( ref . 33 ). we found that cxcl8 ( 3 - 74 ) k11r / g31p was a potent antagonist of cxcl8 binding in this assay ( fig1 ), such that 10 ng / ml of cxcl8 ( 3 - 74 ) k11r / g31p blocked 95 % of subsequent biot cxcl8 binding to the cells . when tested at this dose , cxcl8 ( 3 - 74 ) k11r / p32g blocked only 48 % of cxcl8 binding , while unlabeled cxcl8 itself competitively inhibited 30 % of biot cxcl8 binding . introduction into cxcl8 ( 3 - 74 ) k11r / g31p or cxcl8 ( 3 - 74 ) k11r / p32g of additional amino acid substitutions at thr12 and his13 substantially reduced the antagonist activities of the analogues ( fig1 ). this data clearly suggests that pre - incubation of neutrophils with cxcl8 ( 3 - 74 ) k11r / g31p strongly down - regulates subsequent binding of cxcl8 . in order to more finely map the ability of cxcl8 ( 3 - 74 ) k11r / g31 to inhibit the binding of cxcl8 , in our next set of experiments we simultaneously exposed the cells to 125 icxcl8 and varying doses of cxcl8 ( 3 - 74 ) k11r / g31p or unlabeled cxcl8 . we found that cxcl8 ( 3 - 74 ) k11r / g31p was about two orders of magnitude more effective than wildtype cxcl8 in inhibiting the binding of 20 pm 125 i - cxcl8 to the cells ( fig1 ). the concentration for inhibiting 50 % of labelled ligand binding ( ic 50 ) was 120 pm for unlabelled cxcl8 , and 4 pm for cxcl8 ( 3 - 74 ) k11r / g31p . this data suggests that cxcl8 ( 3 - 74 ) k11r / g31p is a very potent competitive inhibitor of cxcl8 binding to neutrophils . cxcl8 ( 3 - 74 ) k11r / g31p does not display neutrophil agonist activities . while cxcl8 ( 3 - 74 ) k11r / g31p was certainly a high affinity ligand for the neutrophil cxcl8 receptors , it would equally well do so as an agonist or an antagonist . thus our next experiments addressed the potential agonist activities of the cxcl8 ( 3 - 74 ) k11r analogues we generated , as measured by their abilities to chemoattract these cells or induce release of the neutrophil granule hydrolytic enzyme β - glucuronidase in vitro ( fig2 ). we found that even at 100 ng / ml , cxcl8 ( 3 - 74 ) k11r / g31p was a poor chemoattractant , inducing 13 . 9 +/− 4 % or 5 . 4 +/− 2 % of the responses induced by 1 or 100 ng / ml cxcl8 ( p & lt ; 0 . 001 ), respectively . at 100 ng / ml , the cxcl8 ( 3 - 73 ) k11r / p32g analogue induced a response that was fairly substantial ( 38 . 3 +/− 2 % of the cxcl8 response ), while the combined cxcl8 ( 3 - 74 ) k11r / g31p / p32g analogue also was not an effective chemoattractant . when we assessed their abilities to induce - glucuronidase release , we found that none of the cxcl8 ( 3 - 74 ) k11r analogues was as effective as cxcl8 in inducing mediator release . indeed , we found only background release with any of them at 10 ng / ml , and at 100 ng / ml only cxcl8 ( 3 - 74 ) k11r / g31p / p32g induced significant neutrophil responses ( fig2 ). given the combined cxcl8 competitive inhibition and neutrophil agonist data , from this point on we focused our attention on cxcl8 ( 3 - 74 ) k11r / g31p . cxcl8 ( 3 - 74 ) k11r / g31p blocks neutrophil chemotactic responses to both cxcr1 and cxcr2 ligands . the most pathogenic effect of inappropriate elr + chemokine expression is the attraction of inflammatory cells into tissues . thus , we next assessed the impact of cxcl8 ( 3 - 74 ) k11r / g31p on the chemotactic responses of neutrophils to high doses of cxcl8 ( fig3 ). as predicted from our in vivo observations in sheep and cattle ( ref . 33 ), 1 μg / ml ( 129 nm ) cxcl8 was very strongly chemoattractive , but even very low doses of cxcl8 ( 3 - 74 ) k11r / g31p ameliorated this response . the addition of 12 . 9 pm cxcl8 ( 3 - 74 ) k11r / g31p reduced the chemotactic response of the cells by 33 %. the ic 50 for cxcl8 ( 3 - 74 ) k11r / g31p under these conditions was 0 . 11 nm , while complete blocking of this cxcl8 response was achieved with 10 nm cxcl8 ( 3 - 74 ) k11r / g31p . when we tested the efficacy of cxcl8 ( 3 - 74 ) k11r / g31p in blocking responses to more subtle bovine cxcl8 challenges , we also extended the study to assess the ability of cxcl8 ( 3 - 74 ) k11r / g31p to block neutrophil responses to human cxcl8 as well as to the human cxcr2 - specific ligands cxcl1 and cxcl5 . each of these is expressed in the affected tissues of pancreatitis ( ref . 34 ) or ards ( ref . 3 ) patients at 1 – 10 ng / ml . we found that bovine neutrophils were responsive to 1 ng / ml hcxcl1 or hcxcl5 , and similarly responsive to 10 ng / ml hcxcl8 ( fig3 ), so we employed these doses to test the effects of cxcl8 ( 3 - 74 ) k11r / g31p on neutrophil responses of these ligands . the neutrophil responses to hcxcl1 and hcxcl5 were reduced to 50 % by 0 . 26 and 0 . 06 nm cxcl8 ( 3 - 74 ) k11r / g31p , respectively , while their responses to hcxcl8 were 50 % reduced by 0 . 04 nm cxcl8 ( 3 - 74 ) k11r / g31p ( fig3 ). this data indicates that cxcl8 ( 3 - 74 ) k11r / g31p can antagonize the actions of multiple members of the elr - cxc subfamily of chemokines . cxcl8 ( 3 - 74 ) k11r / g31p is an effective in vitro antagonist of the neutrophil chemokines expressed in bacterial pneumonia or mastitis lesions . we wished to test the extent to which our antagonist could block the array of neutrophil chemoattractants expressed within complex inflammatory environments in vivo . thus , we chose two diseases in which chemokine - driven neutrophil activation contributes importantly to the progression of the pathology , mastitis and pneumonic mannheimiosis . we utilized an endotoxin model of mastitis ( ref . 35 ), in which we infused 5 μg of endotoxin / teat cistern and 15 h later lavaged each cistern . neutrophils comprised 82 and 6 %, respectively , of the cells from endotoxin and saline - control cisterns , with the bulk of the remaining cells comprising macrophages . the diluted ( 1 : 10 ) wash fluids induced strong in vitro neutrophil chemotactic responses , and the addition of anti - cxcl8 antibodies to the samples maximally reduced these by 73 +/− 8 % ( fig4 a ), relative to the medium control . on the other hand , the addition of 1 ng / ml of cxcl8 ( 3 - 74 ) k11r / g31p to the samples reduced their chemotactic activity by 97 +/− 3 %. neutrophils also comprised 93 +/− 12 % of the cells recovered from the balf of cattle with advanced pneumonic mannheimiosis . when tested in vitro , these samples too were strongly chemotactic for neutrophils , and the addition of anti - cxcl8 antibodies maximally reduced their neutrophil chemotactic activities by 73 +/− 5 % ( fig4 a ). treatment of these balf samples with 1 or 10 ng / ml of cxcl8 ( 3 - 74 ) k11r / g31p reduced the neutrophil responses by 75 +/− 9 or 93 +/− 9 %, respectively , relative to the medium controls . this data suggests that cxcl8 ( 3 - 74 ) k11r / g31p blocks the actions of cxcl8 and non - cxcl8 chemoattractants in these samples . in order to confirm these observations using an alternate strategy , we next depleted bacterial pneumonia balf samples of cxcl8 using immunoaffinity matrices , then assessed the efficacy of cxcl8 ( 3 - 74 ) k11r / g31p in blocking the residual neutrophil chemotactic activities in the samples ( fig4 b ). the untreated lesional balf samples contained 3 , 215 +/− 275 pg / ml cxcl8 , while the immunoaffmity - absorbed balf contained 24 +/− 17 pg / ml cxcl8 . in this series of experiments the neutrophil response to the cxcl8 - depleted balf samples was 65 . 4 +/− 4 % of their responses to the unabsorbed samples . it is known that cxcl8 can contribute as little as 15 % of the neutrophil chemotactic activities in pneumonic mannheimiosis balf obtained from an array of clinical cases ( ref . 9 ). whereas the cxcl8 depletion treatments were 99 % effective in removing cxcl8 , there remained in these samples substantial amounts of neutrophil chemotactic activities , and the addition of 1 ng / ml cxcl8 ( 3 - 74 ) k11r / g31p fully abrogated their cumulative effects ( fig4 b ). this data unequivocally confirmed that cxcl8 ( 3 - 74 ) k11r / g31p also antagonizes the spectrum of non - il - 8 chemoattractants expressed in these samples . cxcl8 ( 3 - 74 ) k11r / g31p is highly efficacious in blocking endotoxin - induced neutrophilic inflammation in vivo . in our last experiments , we assessed the ability of cxcl8 ( 3 - 74 ) k11r / g31p to block endotoxin - induced inflammatory responses in the skin of cattle , as well as the time - frames over which it was effective . the animals were challenged intradermally with 1 μg bacterial endotoxin 15 h before ( internal positive control response ), or at three different times after , intravenous , subcutaneous or intramuscular injection of cxcl8 ( 3 - 74 ) k11r / g31p ( 75 μg / kg ). thus , punch biopsies of 15 h endotoxin reaction sites were taken 15 min before treatment and at 16 , 48 and 72 h after injection of the antagonist into each animal , and the numbers of infiltrating neutrophils were determined in a blinded fashion for the papillary ( superficial ), intermediate and reticular dermis of each biopsy . prior to the antagonist treatments , strong neutrophilic inflammatory responses were evident at the endotoxin challenge sites in each animal ( fig5 ). within the biopsies , the responses in the papillary dermis were mild in all animals ( data not shown ) and became progressively more marked with increasing skin depth , such that maximal inflammation ( neutrophil infiltration ) was observed around the blood vessels in the reticular dermis ( fig5 a ). following the cxcl8 ( 3 - 74 ) k11r / g31p treatments , the inflammatory responses observed within the 16 h biopsies were 88 – 93 % suppressed , while those in the 48 h biopsies were 57 % ( intravenous ) to 97 % ( intradermal ) suppressed , relative to their respective pretreatment responses . by 72 h post - treatment the effects of the intravenously administered antagonist had worn off , while the endotoxin responses in the intradermally and subcutaneously treated cattle were still 60 % suppressed . this data clearly indicates that cxcl8 ( 3 - 74 ) k11r / g31p is a highly effective antagonist of endotoxin - induced inflammatory responses in vivo , that these effects can last for 2 – 3 days , and that the route of delivery markedly affects the pharmacokinetics of this novel antagonist . we have found that g31 antagonizes also the chemotactic effects of the human elr - cxc chemokines cxcl8 / il - 8 and cxcl5 / ena - 78 on human neutrophils . thus , the chemotactic activities of 0 . 1 to 500 ng / ml of either cxcl8 ( fig6 , left panel ) or cxcl5 / ena - 78 ( fig6 , right panel ) were essentially completely blocked by the addition of 10 ng / ml of our antagonist to the chemotaxis assays . similarly , g31p blocked the chemotactic effects of cxcl8 for cxcr1 / cxcr2 - positive eosinophils . we and others have found that eosinophils from atopic or asthmatic subjects express both elr - cxc chemokine receptors , and are responsive to cxcl8 ( fig7 , left panel ). the chemotactic effects of 100 ng / ml cxcl8 , but not the ccr3 ligand ccl11 / eotaxin , on purified peripheral blood eosinophils of an mildly atopic , non - asthmatic donor (‰ 99 % purity ) were completely abrogated by the addition of 10 ng / ml g31p to the chemotaxis assays ( fig7 , middle panel ). when tested against purified eosinophils from a hypereosinophilic patient ( fig7 , right panel ), g31p was neutralized the responses of these cells to either cxcl8 / il - 8 or cxcl5 / ena - 78 . this data clearly indicates that bovine g31p is an effective antagonist of the bovine elr - cxc chemokines expressed in vivo in response to endotoxin challenge , but also can fully antagonize neutrophil and eosinophil elr - cxc chemokine receptor responses to cxcl8 and cxcl5 , known ligands for both the cxcr1 and cxcr2 . we demonstrated herein that cxcl8 ( 3 - 74 ) k11r / g31p is a high affinity antagonist of multiple elr - cxc chemokines . in vitro , this antagonist effectively blocked all of the neutrophil chemotactic activities expressed in mild to intense inflammatory lesions within two mucosal compartments ( lungs , mammary glands ), and up to 97 % blocked endotoxin - induced inflammatory responses in vivo . we identified cxcl8 as a major chemoattractant in the pneumonia and mastitis samples , but also demonstrated that 35 % of the activity in the bacterial pneumonia samples was due to non - cxcl8 chemoattractants that were also effectively antagonized by cxcl8 ( 3 - 74 ) k11r / g31p . based on studies of inflammatory responses in rodents ( ref . 18 , 19 ), cattle ( ref . 8 ), and humans ( ref . 3 ), it is clear that these samples could contain numerous elr + cxc chemokines ( e . g ., cxcl5 , and cxcl8 ) to which cxcl8 ( 3 - 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