BLTR: the Leukotriene B4 Receptor

BLTR: the Leukotriene B4 Receptor Andrew D. Luster* and Andrew M. Tager Infectious Disease Unit, Massachusetts General Hospital, East 149 13th Street, Charlestown, MA 02129, USA * corresponding author tel: 617-726-5710, fax: 617-726-5411, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23008.

SUMMARY The receptor for leukotriene B4 (LTB4), BLTR, is a member of the G protein-coupled seven transmembrane domain receptor (GPCR) superfamily. This receptor is highly expressed on leukocytes, and mediates LTB4-induced leukocyte chemotaxis and activation. BLTR therefore participates in the recruitment of leukocytes to sites of pathogen invasion as part of the innate immune response, and also in the pathogenesis of inflammatory reactions in which LTB4 has been implicated. BLTR antagonists have successfully reduced leukocyte recruitment and tissue damage in animal models of inflammatory diseases, demonstrating the therapeutic potential of inhibiting BLTR in the treatment of pathologic inflammation.

BACKGROUND

Discovery A binding site for [3H]LTB4 on human neutrophils was initially detected in 1982 with a Kd of 10 nM (Goldman and Goetzl, 1982), 0.46 nM (Lin et al., 1984), and 1.5 nM (Bomalaski and Mong, 1987), and on guinea pig eosinophils with a Kd of 1.4 nM (Ng et al., 1991). The human BLTR was eventually cloned from retinoic acid-differentiated HL-60 cells using a subtraction strategy (Yokomizo et al., 1997). Membrane fractions of COS-7 cells transfected with the cloned sequence demonstrated LTB4 binding with a Kd comparable to that observed in retinoic

acid-differentiated HL-60 cells, and CHO cells stably transfected with the sequence demonstrated LTB4induced increases in intracellular calcium and chemotactic responses, indicating that this sequence encoded the LTB4 receptor, BLTR (Yokomizo et al., 1997). This sequence had been previously cloned using degenerate PCR strategies by two independent groups as an orphan receptor gene believed to encode a member of the G protein-coupled seven transmembrane domain receptor (GPCR) superfamily, and called R2 (Raport et al., 1996) and chemoattractant receptor-like 1 (CMKRL1) (Owman et al., 1996). LTB4 was subsequently confirmed to be the functional ligand for this putative receptor in experiments demonstrating high-affinity LTB4 binding and LTB4induced increases in intracellular calcium in cells transfected with CMKRL1 cDNA (Owman et al., 1997). The identical receptor sequence was also isolated from a low stringency screen of a human erythroleukemia (HEL) cell cDNA library using chicken purinoceptor P2Y3 cDNA, and designated P2Y7 based on the ability of the transfected receptor to bind [35S]dATP (Akbar et al., 1996). However, ATP binding and signaling have not been seen by others (Yokomizo et al., 1997; Huang et al., 1998). The mouse ortholog of BLTR was independently cloned by performing degenerate PCR with primers directed to well-conserved transmembrane domains of chemoattractant GPRCs, using cDNA isolated from murine eosinophils (Huang et al., 1998). CHO cells transfected with this mouse sequence bound LTB4 with high affinity and demonstrated LTB4induced increases in intracellular calcium (Huang et al., 1998). Another group subsequently identified

2242 Andrew D. Luster and Andrew M. Tager the identical murine sequence by screening a mouse genomic library with a fragment of the human cDNA identified previously as encoding the P2Y7 receptor (Martin et al., 1999).

Alternative names The consensus nomenclature for this receptor is BLTR (Alexander and Peters, 1997). Prior to its identification as the LTB4 receptor, however, groups cloning this sequence called it R2 (Raport et al., 1996), CMKRL1 (Owman et al., 1996), and P2Y7 (Akbar et al., 1996).

Structure Kyte-Doolittle hydrophobicity analysis of the amino acid sequences of human (Raport et al., 1996; Owman et al., 1996) and mouse (Huang et al., 1998) BLTR shows the presence of seven hydrophobic transmembrane domains common to GPCRs. The amino acid sequences also contain other motifs characteristic of this family of receptors (Owman et al., 1996; Huang et al., 1998), including (a) conserved proline residues in several of the transmembrane domains, which are thought to induce flexibility in the helix formations; (b) conserved cysteine residues in two of the extracellular loops for intramolecular chain disulfide bonding; (c) consensus sequences for N-linked glycosylation near the N-terminus and in one of the extracellular loops; and (d) a serine and threoninerich C-terminal intracytoplasmic segment, which in other GPCRs are sites of phosphorylation involved with receptor desensitization and internalization (Murphy, 1994). Additionally, human BLTR is 352 amino acid residues in length, and mouse BLTR is 351 amino acids, similar to the lengths of other chemoattractant receptors (Murphy, 1994).

Main activities and pathophysiological roles LTB4 was discovered based on its potent chemotactic activity on neutrophils (Ford-Hutchinson et al., 1980), and BLTR probably mediates this effect: CHO cells stably expressing exogenous BLTR showed marked chemotactic responses towards low concentrations of LTB4 (Yokomizo et al., 1997). In addition to chemotaxis, LTB4 stimulates neutrophil aggregation, adherence associated with increased CD11/ CD18 expression, generation of reactive oxygen species and release of granular enzymes (Henderson, 1994).

In addition to its effects on neutrophils, LTB4 stimulates chemotaxis of guinea pig eosinophils (Ng et al., 1991) and IL-5-exposed murine eosinophils (Huang et al., 1998). Human eosinophils, however, have been reported to respond poorly to LTB4 when compared with PAF (Morita et al., 1989). LTB4 augments human peripheral blood monocyte production of IL-1 (Rola-Pleszczynski and Lemaire, 1985), and mouse peritoneal macrophage phagocytosis and killing of bacteria (Demitsu et al., 1989). Mice with targeted disruption of the 5-lipoxygenase (5-LO) gene, and hence deficient of leukotrienes including LTB4, showed a greater degree of lethality as well as bacteremia following intratracheal challenge with Klebsiella pneumoniae (Bailie et al., 1996). Alveolar macrophages from the 5-LO-deficient mice exhibited impairments in phagocytosis and killing of bacteria in vitro, which were overcome by addition of exogenous LTB4. LTB4 enhances production of IL-2 by CD4+ T cells (Marcinkiewicz et al., 1997), and IL-2R expression by CD8+ T cells (Stankova et al., 1992). LTB4 increases NK cell IL-2R expression, sensitivity to IL-2, and cytotoxic activity (Rola-Pleszczynski et al., 1983; Stankova et al., 1992). LTB4 enhances B cell activation, proliferation, and Ig secretion (Yamaoka et al., 1989). Treatment of endothelial cell monolayers with LTB4 increases their binding of neutrophils (Gimbrone et al., 1984). LTB4 treatment of endothelial cells has also been reported to increase binding of lymphocytes (Renkonen et al., 1988), but this finding was not substantiated in a subsequent study (To and Scrieber, 1990). Inflammatory Cell Recruitment By mediating LTB4-induced leukocyte chemotaxis and activation, BLTR participates in the recruitment of leukocytes to sites of pathogen invasion as part of the innate immune response. By mediating LTB4induced leukocyte chemotaxis and activation, BLTR also participates in the pathogenesis of inflammatory diseases in which LTB4 has been implicated, including asthma and allergic rhinitis, cystic fibrosis, inflammatory bowel disease, psoriasis, acute respiratory distress syndrome, multiple sclerosis, and rheumatoid arthritis (Samuelsson et al., 1987; Lewis et al., 1990; Henderson, 1994). HIV Coreceptor Some members of the GPCR superfamily of chemoattractant receptors, including CCR5 and CXCR4, also function as coreceptors for HIV entry into target cells. The ability of other members of this family,

BLTR: the Leukotriene B4 Receptor 2243 including BLTR, to serve as HIV coreceptors has recently been investigated, with conflicting results. One group demonstrated that BLTR was able to act as a coreceptor for entry of several clinical isolates of HIV-1 into murine fibroblasts stably expressing both BLTR and the human CD4 receptor (Owman et al., 1998). Env proteins of these clinical isolates were not described. However, a second group subsequently found that BLTR did not function as a coreceptor for either Env-mediated viral entry or Env-mediated cell± cell fusion in experiments using various HIV-1 and SIV Env proteins (Martin et al., 1999). Figure 1 Human 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001

BLTR gccattctct gctgcggttt ggcttttctc ccattatact ggtcagattg tctgtcattc gggttcacat ctacacccag tggtctcaaa taccggtatg ttcaaggaaa ctggtttccc gctcctcatg tagtagctgt cagactcagc taattggcat atggagaatt gctggaagat agtgctgccc caaacccaga aaagcgaaac ttctctggct ccagtctaga cctctctcac tgccttctgg gggtttcagc ctctctggct agatccaggg cagttcctgc acccccctca gctggctgtg gaagcgctct gctcactgct tggttgccgc cacggccatg acgcaccaag ggccacaccc cttcccgcgg gggcttcctg acaggcccgg gaccttcgcc gctggccggc cgtgctcatc cggcggcggc gggttccgag ccccgccgct aaacgaactg tctgagccag agcgtgggag gtgagagcgt aa

GENE

Accession numbers Human: NM000752, X98356, U41070, U33448 Mouse: AF044030

Sequence See Figure 1.

Nucleotide sequence of the human BLTR gene.

cacatcccgt ctgcccattt caagcagaac ttgcatcttt aaggaaggac aggctggagt gattctcctg ctaacttttg ctgctaacat aaccaccaca ggagacattc ctcttggcag ctcagcctgc gagaagatag agggtaacca tctgtgtgat ggttgaccta ggccctcttc ccttcggcat cccaggacct aaacacaaaa accatacctc cctgcatctt tgctccttcc gttgccctgg cccaccctca gatttagcaa ataggctaca caaagcttgt ctaggtgtag gggcttcccg gtcactgccc ccctttttcc ctgtgtcact agtctagacc gcgatggccc gtcctcgcgt taccccagcg ctgcccttcc cgcttccgcc gccttctggc caggccgccg gcactcgcct ctgctgcgct gcgtccagca ctggagcccg aactaggcct ttcagtacct gcgggagtgg gctccagcct

gcggtcagga ttttcatatc aagtgggggc cctgagaagt tttttagttt gcagtggtgc cctcagcctc tatttttagt caagtgatct acctgccagg ctctgtccag ggtgggcatg cagctcggcc gaaagaggta catgggcaag accaaggaga aatggaacca ccaccaccta ttactgatgt agtcacagct agatatcagc cttctcctat gaggccacac tggtctcttc aaaacagact ggaagatgcg acagcaccta ccaccctgcc aagtcctccc agttcatctc gcaacagctt tgatggtgct ttcacttcct atgtctgcgg gctcactggc ggcgggtgct accgcacagt aagggcaccg tggctgtggt gcagccgccg tgccctacca ggttagggct tcctgagcag cggcgggcgt cgcgccgcgg gcccttccga ggtggaagga ggaggaggag agtggaagaa ggctcccaca

agcccttcct ctctgacagc tctggaaagg gagagttgaa cttttttttt gatctcagct ccaagtagct agagacgggg gctcccctca aatttttagt gaaacgggta aggcatcact tcaactttgt gtgccaatct cacaaggtag ccatttggat gtccctttaa gatcatagct gccctcttta ccaacctaca attgtagcct atgataccat ccagccttct tcatctggcc atcccccctc tcttccctgt gacctggggc ctgaccctgg gacggccatg tctgctggct tgtggtgtgg gaacctggcc ggcccaaggc agtcagcatg ggtggcccgc ggcaggcatc agtgccctgg ggccttccat ggccagctac caccggccgc cgtggtgaac cgtggggaag cagcgtgaac gggcttcgtc gggcagcctg gagcctcact ggcgcacttt caggggcgtg gagggagaga ggcagcttta

gaactctgac tgcgaggtca ttaagggacc agggaagcag tttttgaaat cactgcagcc gagactacag tttcaccatg gcctcccaaa ttttagcttt aggggaccat gttcctgctc gtgtctaaag ccttgcccag gtgcttgggg tttggcttct gtaaggggag tgaactgaag aatcatgatg cttcctatta ccaatctgag tcactacttt cactccccac ccacctctaa ctagtgaagg cctctgctct caggcctttg gattggcatc aacactacat atcatcctgc agtatcctga ctggccgacc acctggagtt tacgccagcg ccctttgtgt tgggtgttgt aaaacgaaca ctaatcttcg tcggacatag ctggtggtgc ctggctgagg cggctgagcc cccgtgctgt gccaagctgc ggccagaccg gcctccagcc cctcctggca gagggcgtgg tggagcaaag accattaaaa

ttcagttctt tctctgctct tcagtggcca gaaggcccat ggagtctcgc tccacttcct gcacatgcca ttggccaggc gtgctgggat tgcaggagac ttctgcattg cctcactcct tggaactgaa atcataaatc aaaggggaag accaaagaga gaaagggggt ccaaggacag ttatctaacc atcttaaaac cccatttccc gttcaattat acccctcttt ggagtcctcc gagtgggtag gtggtacttc gcagtgggac agcttccaac cttctgcagc tgtcagtggc aaaggatgca tggccgtatt ttggactggc tcctgcttat cccagaagct cctttctgct tgagcctgtg aggctgtcac ggcgtcggct tcatcatcct cgggccgcgc tggcccgcaa acgcgtgcgc tggagggcac ctaggagcgg ctctcaagtt gaatgctagc agggcgtggg tgagggccga ctgaagtctg

2244 Andrew D. Luster and Andrew M. Tager

Chromosome location and linkages When human BLTR was cloned as R2, CMKRL1, and P2Y7, it was localized to chromosome 14. Probing Southern blots of mouse/human somatic cell hybrid DNA with R2 cDNA localized the gene to the region 14pter-14q23 (Raport et al., 1996). Mapping by FISH/DAPI using CMKRL1 cDNA showed the gene localized to chromosome 14q11.2-q12 (Owman et al., 1996). Mapping with PCR using DNA from a panel of mouse/human somatic cell hybrids as templates and primers from the P2Y7 cDNA clone produced a similar result: amplification of the expected size product was observed only with DNA from the hybrid that contained human chromosome 14 (Akbar et al., 1996). Human somatostatin receptor 1 (SSTR1), a member of the GPCR superfamily, has been mapped to chromosome 14q13 (Yamada et al., 1993), close to the location of BLTR on chromosome 14q11.2-q12 (Owman et al., 1996).

PROTEIN

Relevant homologies and species differences The human proteins with the highest overall homology to human BLTR are the human somatostatin receptor type 5 (SSTR5), with 33.0% identity; the human IL-8 receptor (CXCR1), with 33.0%; the human formyl peptide-related receptor II (FMLPRII, the lipoxin A4 receptor), with 30.7%; and the human formyl peptide receptor (FMLPR), with 28.6% (Yokomizo et al., 1997). Mouse BLTR, which has 78% identity with human BLTR, has 32% identity with the murine C5a receptor (mC5aR), 32% identity with the murine IL-8 receptor (mCXCR2), 30% identity with murine FMLPR, 29% identity with the murine lipoxin A4 receptor (mLXAR), and 26% identity with the murine platelet-activating factor receptor (mPAFR) (Huang et al., 1998). Beyond the overall 78% identity between human and mouse BLTR, the three intracytoplasmic loops are identical across these species, while not being conserved across the subfamily of chemoattractant receptors, suggesting that the BLTRs may be coupled to a unique, well-conserved, signaling pathway among the chemoattractant receptors (Huang et al., 1998).

Accession numbers Human: NP000743, AAB16747 Mouse: AAC61677

CAA67001,

AAC50628,

Affinity for ligand(s) Cells transfected with human and mouse BLTR demonstrate LTB4 binding with Kd values comparable to that observed with leukocytes (Yokomizo et al., 1997; Huang et al., 1998). Although the human BLTR sequence was reported to bind ATP with high affinity, exposure to ATP induced no changes in

Sequence See Figure 2.

Figure 2 Amino acid sequences of human and mouse BLTR. Human 1 61 121 181 241 301

BLTR: MNTTSSAAPP ALADLAVLLT RPFVSQKLRT HLIFEAVTGF NLAEAGRALA VAKLLEGTGS

SLGVEFISLL APFFLHFLAQ KAMARRVLAG LLPFLAVVAS GQAAGLGLVG EASSTRRGGS

AIILLSVALA GTWSFGLAGC IWVLSFLLAT YSDIGRRLQA KRLSLARNVL LGQTARSGPA

VGLPGNSFVV RLCHYVCGVS PVLAYRTVVP RRFRRSRRTG IALAFLSSSV ALEPGPSESL

WSILKRMQKR MYASVLLITA WKTNMSLCFP RLVVLIILTF NPVLYACAGG TASSPLKLNE

SVTALMVLNL MSLDRSLAVA RYPSEGHRAF AAFWLPYHVV GLLRSAGVGF LN

Mouse 1 61 121 181 241 301

BLTR: MAANTTSPAA NLALADLAVL VARPFMSQKV FHLLFEAITG VNLVEAGRTV VVKLLEGTGS

PSSPGGMSLS LTAPFFLHFL RTKAFARWVL FLLPFLAVVA AGWDKNSPAG EVSSTRRGGT

LLPIVLLSVA ARGTWSFREM AGIWVVSFLL SYSDIGRRLQ QRLRLARYVL LVQTPKDTPA

LAVGLPGNSF GCRLCHYVCG AIPVLVYRTV ARRFRRSRRT IALAFLSSSV CPEPGPTDSF

VVWSILKRMQ ISMYASVLLI KWNNRTLICA GRLVVLIILA NPVLYACAGG MTSSTIPESS

KRTVTALLVL TIMSLDRSLA PNYPNKEHKV FAAFWLPYHL GLLRSAGVGF K

BLTR: the Leukotriene B4 Receptor 2245 intracellular calcium in cells transfected with the human BLTR (Yokomizo et al., 1997), and there was no detectable specific binding of ATP to cells transfected with mouse BLTR (Huang et al., 1998).

Cell types and tissues expressing the receptor Northern blotting of various human tissues with human BLTR cDNA demonstrated that gene expression is highest in peripheral blood leukocytes, and also present in spleen, thymus, bone marrow, lymph nodes, heart, skeletal muscle, brain, and liver (Akbar et al., 1996; Owman et al., 1996; Yokomizo et al., 1997). Northern blotting with mouse BLTR cDNA revealed gene expression predominantly in activated leukocytes, including IL-5-exposed eosinophils, elicited peritoneal neutrophils and macrophages, and IFN -stimulated macrophages. Additionally, mouse BLTR was highly expressed in T cell lymphomas that spontaneously arose in c-myc transgenic mice homozygous for p53-null alleles, suggesting that T cells can express BLTR. Low levels of constitutive expression were also seen in the lung, lymph nodes, and spleen (Huang et al., 1998). Increased expression of BLTR mRNA was demonstrated in the lungs of mice subjected to inhalation of the mold allergen Aspergillus fumigatus, consistent with the induction of an intense eosinophil-predominant inflammatory infiltrate (Huang et al., 1998). Using an affinity-purified rabbit antibody directed against the N-terminal 17 amino acids of the predicted open reading frame of mouse BLTR (Huang et al., 1998), expression of this protein was demonstrated by western blotting in eosinophils purified from IL-5 transgenic mice and alveolar macrophages isolated by bronchoalveolar lavage (BAL) from wildtype mice. The antibody crossreacts with human BLTR, and expression of the human protein was demonstrated in human eosinophils (Huang et al., 1998).

Regulation of receptor expression Mouse BLTR transcription is induced in the RAW 264.7 macrophage cell line by IFN . Mouse BLTR transcription, which is not detectable in resting peritoneal cells, is dramatically induced in both activated macrophages and neutrophils elicited into the peritoneum of mice by injections of 9% sodium casein (Huang et al., 1998).

One group cloning the mouse BLTR reported putative binding sites for several transcription factors upstream from the gene's open reading frame (Martin et al., 1999). However, analysis of murine BLTR cDNA (Huang et al., 1998) demonstrates the presence of a small untranslated exon upstream of the region examined by this group, indicating that these putative binding sites are actually in an intron of the gene (unpublished observations). Neither the human nor mouse BLTR promoters have as yet been identified. Mouse BLTR has been demonstrated to be Nlinked glycosylated. In vitro translation of the cDNA in the presence of dog pancreatic microsomes revealed an upward shift in mobility of the protein product on SDS-PAGE of  4 kDa, compared with the protein product translated in the absence of microsomes (Huang et al., 1998).

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades BLTR is a G protein-coupled receptor. LTB4 cytoplasmic signaling has been shown to be predominantly mediated by Bordetella pertussis toxin (PTX)-sensitive G protein(s) leading to the activation of phosphoinositide (PI)-specific phospholipase C, release of inositol phosphates through PI hydrolysis and subsequent mobilization of intracellular calcium (Gaudreau et al., 1998). However, experiments using CHO cells stably expressing BLTR demonstrated that whereas LTB4 induced inhibition of forskolininduced adenylyl cyclase activity was completely blocked by PTX, LTB4-induced increases in intracellular calcium concentration and D-myo-inositol-1,4,5trisphosphate (IP3) accumulation were only partially blocked by PTX, indicating that BLTR couples to both PTX-sensitive and insensitive G proteins in BLTR transfected CHO cells (Yokomizo et al., 1997). Similarly, LTB4-induced increases in intracellular calcium in leukocytes is PTX-sensitive (Powell et al., 1996), whereas LTB4 induced increases in the adhesiveness of vascular endothelial cells for leukocytes is not (Palmblad et al., 1994), suggesting that BLTR couples to different types of G proteins in different cell types (Yokomizo et al., 1997). Whereas sensitivity to PTX suggests involvement of Gi/0 subunits in BLTR signaling, experiments using a cotransfection system in COS-7 cells indicate that this receptor can couple to a PTX-insensitive  subunit of the Gq class of G proteins, specifically G16 (Gaudreau et al., 1998).

2246 Andrew D. Luster and Andrew M. Tager

Transgenic mice overexpressing human BLTR demonstrate markedly amplified neutrophil recruitment and 5-lipooxygenase signaling in murine models of acute skin inflammation, peritonitis, and reperfusioninitiated second organ injury (Chiang et al., 1999). The phenotype of BLTR knockout mice has not yet been described.

treatment of mice with a selective LTB4 receptor antagonist inhibited neutrophil influx into the colonic mucosa (Fretland et al., 1995). In a murine model of experimental allergic encephalitis, pretreatment of mice with a selective LTB4 receptor antagonist markedly blocked the recruitment of eosinophils into the spinal cord and completely inhibited the development of paralysis (Gladue et al., 1996). Administration of a selective LTB4 receptor antagonist markedly blocked the massive influx of inflammatory cells in the subsynovial connective tissue in murine collageninduced arthritis, and abrogated the destruction of articular cartilage and erosion of bone (Showell et al., 1998). Administration of selective LTB4 receptor antagonists have also been shown to significantly prolong allograft survival in a rat model of liver transplantation (Ii et al., 1996), and a murine cardiac allograft model (Weringer et al., 1999). Finally, in a murine model of acute septic peritonitis, administration of a selective LTB4 receptor antagonist inhibited recruitment of both neutrophils and macrophages, and led to significantly increased mortality (Matsukawa et al., 1999). A selective LTB4 receptor antagonist was administered to human asthma patients prior to whole lung allergen challenge in one double-blind placebocontrolled crossover trial (Evans et al., 1996). The receptor antagonist significantly reduced the number of neutrophils recruited into BAL fluid, although this was not associated with measurable physiological benefit. A randomized controlled trial of a selective LTB4 receptor antagonist in patients with psoriasis demonstrated no statistically significant differences in median psoriasis area or severity index between treatment groups (Van Pelt et al., 1998).

THERAPEUTIC UTILITY

References

Effects of inhibitors (antibodies) to receptors

Akbar, G., Dasari, V., Webb, T., Ayyanathan, K., Pillarisetti, K., Sandhu, A., Athwal, R., Daniel, J., Ashby, B., Barnard, E., and Kunapuli, S. (1996). Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells. J. Biol. Chem. 271, 18363±18367. Alexander, S. P. H., and Peters, J. A. (1997). Receptors and ion channel nomenclature. Trends Pharmacol. Sci. suppl. 45. Bailie, M. B., Standiford, T. J., Laichalk, L. L., Coffey, M. J., Strieter, R., and Peters-Golden, M. (1996). Leukotrienedeficient mice manifest enhanced lethality from klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities. J. Immunol. 157, 5221± 5224. Bomalaski, J. S., and Mong, S. (1987). Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors. Prostaglandins 33, 855±867. Chiang, N., Gronert, K., Clish, C. B., O'Brien, J. A., Freeman, M. W., and Serhan, C. N. (1999). Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors BLTR has been demonstrated to mediate LTB4induced chemotaxis: CHO cells stably expressing human BLTR showed marked chemotactic responses towards low concentrations of LTB4 (Yokomizo et al., 1997). In the stable CHO cell transfectants, activation of BLTR with LTB4 also induced rapid increases in intracellular calcium concentration and IP3 accumulation, as well as inhibition of forskolin-stimulated adenylyl cyclase activity (Yokomizo et al., 1997). LTB4 also induced a dose-dependent intracellular calcium flux in CHO cells stably expressing mouse BLTR (Huang et al., 1998).

Phenotypes of receptor knockouts and receptor overexpression mice

Several selective BLTR antagonists have been developed, and used to block the actions of LTB4 in cell culture, in animal studies, and in human subjects. BLTR antagonists are able to block specific binding of radiolabeled LTB4 to leukocytes in vitro, as well as inhibit various cell functions activated by LTB4, including chemotaxis (Showell et al., 1998; Jackson et al., 1999). Administration of specific BLTR antagonists have inhibited recruitment of leukocytes into tissues in vivo in several animal models of inflammatory diseases. In a murine model of inflammatory bowel disease,

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