Experiments using electrical and mechanical stimulation of the dura in animals with no history of head pain have been used to elucidate the mechanisms of the trigeminovascular system at the level of the trigeminal ganglion, trigeminal nucleus caudalis, periaqueductal gray, and the thalamus. The limitation of these acute models is that electrical and mechanical stimulation of the dural blood vessels does not cause long lasting pain similar to recurrent headache pain in humans. Two animal models for the investigation of headache pathophysiology that include aspects of the chronic nature of headache were included in this study. These models provide a new approach to study the pathophysiology of recurrent headache and support validation of novel treatments.

The results of investigations in these laboratories of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines as human CCR5 antagonists have recently been disclosed. To facilitate further development of these antagonists, we have developed a pharmacophore model based on the structure-activity relationships (SAR) and a human CCR5 receptor docking model using the crystal structure of rhodopsin as a template [Palczewski, K., et al. (2000) Science 289, 739-745]. Guided by the receptor docking model, we have mapped the compounds' site of interaction with CCR5 using site-directed mutagenesis experiments. Our results are consistent with a binding site for the two series that is located within a cavity near the extracellular surface formed by transmembrane helices 2, 3, 6, and 7. This site is overlapping yet distinct from that reported for another antiviral agent which binds to CCR5 [Dragic, T., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 5639-5644].

Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The beta-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop in vivo models of inflammatory diseases, it is essential to identify and characterize the homologues of human eotaxin (C-C chemokine ligand 11) and CCR3 from other species, such as non-human primates. Accordingly, we cloned the macaque eotaxin and CCR3 genes and revealed that they were 91 and 92% identical at the amino acid level to their human homologues, respectively. Macaque CCR3 expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (K(d) = 0.1 nm) and exhibited a robust eotaxin-induced Ca(2+) flux and chemotaxis. Characterization of beta-chemokines on native macaque CCR3 on eosinophils was performed by means of eotaxin-induced shape change in whole blood using a novel signaling assay known as gated autofluorescence forward scatter. Additionally, mAbs were raised against macaque CCR3 using two different immunogens: a 30-amino acid synthetic peptide derived from the predicted NH(2) terminus of macaque CCR3 and intact macaque CCR3-transfected cells. These anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist activity in receptor binding and functional assays. The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of ameliorating chronic inflammatory diseases in humans.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

(2S)-2-(3,4-Dichlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (3) has been identified as a potent CCR5 antagonist lead structure having an IC50 = 35 nM. Herein, we describe the structure-activity relationship studies directed toward the requirement for and optimization of the C-2 phenyl fragment. The phenyl was found to be important for CCR5 antagonism and substitution was limited to small moieties at the 3-position (13 and 16: X= H, 3-F, 3-Cl, 3-Me).

ABSTRACTThe chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in smalltrans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.

The alpha chemokine receptor CXCR4 and its only characterized chemokine ligand, stromal cell-derived factor-1 (SDF-1), are postulated to be important in the development of the B-cell arm of the immune system. In addition, CXCR4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants which predominate in some infected individuals in end stage disease. SDF-1 can block X4-tropic HIV-1 infection of CD4+ target cells in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Therefore, CXCR4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS), and knowledge of the pharmacology of SDF-1 binding to its cognate receptor will be important in the interpretation of these experiments. We report here a Kd derived using a competition binding assay of 4.5 nM for CXCR4 endogenously expressed on peripheral blood monocytes and T-cells. This affinity is similar to that which SDF-1 exhibits when binding to endogenous CXCR4 on an established immortal Jurkat T-cell line as well as recombinant CXCR4 transfected into Chinese Hamster Ovary (CHO) cells. We also demonstrate that the determined affinity of SDF-1 for CXCR4 is reflective of its ability to induce a CXCR4-mediated signal transduction in these different cell types. Furthermore, using Bordetella pertussis toxin, we observe that high affinity binding of SDF-1 to CXCR4 is independent of the G-protein coupled state of the receptor, as uncoupling of G-protein did not lead to the appearance of measurable low affinity SDF-1 binding sites. Moreover, binding affinity and receptor number were unaffected by uncoupling for both recombinant and endogenously expressed CXCR4. Thus, SDF-1 is novel among agonist ligands of G protein-coupled receptors in that it appears to have equal affinity for both the G protein-coupled and uncoupled states of CXCR4.

Knowledge of a receptor' pharmacology provides information which is often essential to unraveling the biological function of that receptor. The chemokine system represents a striking example as most receptors in the family bind multiple chemokines, often with complex overlapping selectivities. Two basic methodologies are used to characterize the binding properties of a receptor. In saturation protocols, the binding of a given ligand, usually labeled to allow detection, is determined over a range of concentrations appropriate for that ligand. Competition experiments measure the ability of varying concentrations of an unlabeled ligand to inhibit the binding of a fixed concentration of a labeled ligand. Although saturation protocols have the advantage of theoretical simplicity, they have three significant disadvantages:KeywordsHank Balance Salt SolutionIncubation BufferReceptor ConcentrationFilter PlateNonspecific BackgroundThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

IP10 and MIG are two members of the CXC branch of the chemokine superfamily whose expression is dramatically up-regulated by interferon (lFN)-gamma, The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated in mediating some of the effects of IFN-gamma and Lipopolysaccharides (LPSs), as well as T-cell-dependent anti-tumor responses. Recently both chemokines have been shown to be functional agonists of the same G-protein-coupled receptor, CXCR3, We now report the pharmacological characterization of CXCR3 and find that, when heterologously expressed, CXCR3 binds IP10 and MIG with K-i values of 0.14 and 4.9 nm, respectively. The receptor has very modest affinity for SDF-1 alpha and little or no affinity for other CXC-chemokines, The properties of the endogenous receptor expressed on activated T-cells are similar. Surprisingly, several CC-chemokines, particularly eotaxin and MCP-4, also compete with moderate affinity for the binding of IP10 to CXCR3, Eotaxin does not activate CXCR3 but, in CXCR3-transfected cells, can block IP10-mediated receptor activation. Eotaxin, therefore, may be a natural CXCR3 antagonist.

Eotaxin is a newly discovered C-C chemokine which preferentially attracts and activates eosinophil leukocytes by acting specifically on its receptor CCR3. The airway inflammation characteristic of asthma is believed to be, at least in part, the result of eosinophil-dependent tissue injury. This study was designed to determine whether there is increased expression of eotaxin and CCR3 in the bronchial mucosa of asthmatics and whether this is associated with disease severity. The major sources of eotaxin and CCR3 mRNA were determined by co-localization experiments. Bronchial mucosal biopsy samples were obtained from atopic asthmatics and normal non-atopic controls. Eotaxin and CCR3 mRNA were identified in tissue sections by in situ hybridization (ISH) using radiolabeled riboprobes and their protein product visualized by immunohistochemistry (IHC). Co-localization experiments were performed by double ISH/IHC. Eotaxin and CCR3 (mRNA and protein) were significantly elevated in atopic asthmatics compared with normal controls. In the asthmatics there was a highly significant inverse correlation between eotaxin mRNA+ cells and the histamine provocative concentration causing a 20% fall in FEV1 (PC20). Cytokeratin-positive epithelial cells and CD31+ endothelial cells were the major source of eotaxin mRNA whereas CCR3 co-localized predominantly to eosinophils. These data are consistent with the hypothesis that damage to the bronchial mucosa in asthma involves secretion of eotaxin by epithelial and endothelial cells resulting in eosinophil infiltration mediated via CCR3. Since selective (eotaxin) and non-selective C-C chemokines such as RANTES, MCP-3 and MCP-4 all stimulate eosinophils via CCR3, this receptor is potentially a prime therapeutic target in the spectrum of diseases involving eosinophil-mediated tissue damage.

C–C chemokine receptor 2 (CCR2) is one of the key players involved in the transmigration of mononuclear cells into the central nervous system (CNS) and subsequent development of multiple sclerosis (MS). The aim of the current study was to analyse the association of CCR2 + 190 G/A (rs1799864) polymorphism with susceptibility to MS and its influence on the age at onset, severity and neurological disability in MS. CCR2 genotyping was carried out by a polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) in 301 MS patients and 342 healthy controls. Logistic regression analysis suggested a marginally significant association between MS and rs1799864 A allele (AA + GA vs. GG, P = 0.047, OR = 1.50, 95% CI = 1.00–2.25), however, after stratification of study groups for the presence of HLA-DRB1*15:01 risk allele, this association could be found in HLA-DRB1*15:01-negative individuals only (AA + GA vs. GG, P = 0.014, OR = 1.84, 95% CI = 1.13–2.98). Furthermore, there was no association between CCR2 polymorphism and clinical features of MS. In conclusion, our results suggest that CCR2 + 190 G/A polymorphism may increase the susceptibility to MS, but its action seems to be restricted to individuals who do not possess the major risk allele HLA-DRB1*15:01.

Although there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of beta-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and RANTES with K(d)s of 0.1, 2.7, and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-1 alpha or MIP-1 beta. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a Ca2+-flux. Competition binding studies on primary eosinophils gave binding affinities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-1 alpha.

The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.

The variable heavy and light chain genes of a monoclonal, IgM, anti-MAG antibody from a patient with neuropathy were inserted into expression vectors containing the gamma and kappa constant regions respectively and co-transfected into monkey kidney CV1P cells. The expressed antibody had the same antigenic specificity but significantly lower avidity than the native IgM, anti-MAG, antibody as detected by ELISA. When the variable heavy chain gene of the anti-MAG antibody was co-transfected with the variable light chain gene from another monoclonal, IgM, anti-MAG antibody, a fully assembled antibody was expressed as determined by a trapping ELISA, but it did not bind to (MAG) or to sulfated glucuronic acid paragloboside, indicating that both heavy and light chains contribute to the binding activity.

The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G --> A transition at bp 867 encoding an arginine --> tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine --> tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4+ protein.