Stefen A. Boehme

The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia.

Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and 3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS.

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Anti-sexual and anxiogenic behavioral consequences of corticotropin-releasing factor overexpression are centrally mediated

Corticotropin-releasing factor (CRF) acts as a neurotransmitter in brain to promote behavioral responses such as flight and immobility, which have adaptive value in the context of exposure to environmental stressors. CRF also suppresses behavioral repertoires such as mating, which are incompatible with such threat-related coping responses. In this study, we employed transgenic (Tg) mice which overexpress CRF in brain and exhibit a constitutive and persistent phenotype of emotionality in order to determine the consequences of long-term CRF excess on indices of reproductive success, male sexual performance and female sexual receptivity. Sexual performance of CRF Tg males was relatively intact, whereas female receptivity was masked in CRF Tg mice by active rejection of sexually experienced male counterparts. This impairment in social interaction was only partially normalized by the serotonin antagonist, methysergide, which enhanced olfactory exploration of the still non-receptive CRF Tg females. Moreover, the anxiogenic-like character of CRF Tg mice is likely to be centrally mediated, since attenuation of hypercorticosteronemia by adrenalectomy did not alter either impaired sexual receptivity or fear-like behavior in an animal model of anxiety. Thus, overexpression of CRF in the brain results in a variety of adverse consequences including diminished social interactions.

Cutting Edge: Serotonin Is a Chemotactic Factor for Eosinophils and Functions Additively with Eotaxin

Elevated levels of serotonin (5-hydroxytryptamine, 5-HT) are observed in the serum of asthmatics. Herein, we demonstrate that 5-HT functions independently as an eosinophil chemoattractant that acts additively with eotaxin. 5-HT2A receptor antagonists (including MDL-100907 and cyproheptadine (CYP)) were found to inhibit 5-HT-induced, but not eotaxin-induced migration. Intravital microscopy studies revealed that eosinophils roll in response to 5-HT in venules under conditions of physiological shear stress, which could be blocked by pretreating eosinophils with CYP. OVA-induced pulmonary eosinophilia in wild-type mice was significantly inhibited using CYP alone and maximally in combination with a CCR3 receptor antagonist. Interestingly, OVA-induced pulmonary eosinophilia in eotaxin-knockout (Eot−/−) mice was inhibited by treatment with the 5-HT2A but not CCR3 receptor antagonist. These results suggest that 5-HT is a potent eosinophil-active chemoattractant that can function additively with eotaxin and a dual CCR3/5-HT2A receptor antagonist may be more effective in blocking allergen-induced eosinophil recruitment.

CRTH2 antagonism significantly ameliorates airway hyperreactivity and downregulates inflammation-induced genes in a mouse model of airway inflammation

Prostaglandin D(2), the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD(2) and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD(2)-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation.

MIP-3ALPHA INDUCES HUMAN EOSINOPHIL MIGRATION AND ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN KINASES (P42/P44 MAPK)

The CC chemokine macrophage inflammatory protein‐3α (MIP‐3α) is the product of recent electronic cloning efforts, however, little characterization of its spectrum of biological effects has been undertaken. Human eosinophils exhibited pertussis‐toxin‐sensitive migration in response to human recombinant (hr)MIP‐3α. Messenger RNA for the MIP‐3α receptor, CCR‐6, and low levels of surface expression were demonstrated by reverse transcriptase‐polymerase chain reaction and FACS analysis. Analyses of cell signaling revealed dose‐dependent increases in intracellular calcium mobilization, calcium transients that were, however, greatly reduced when compared with MCP‐3‐induced responses. Further investigations of MIP‐3α‐induced signal transduction revealed time‐ and dose‐dependent, partially pertussis toxin‐dependent, increases in phosphorylation of the p42/p44 mitogen‐activated protein kinases (MAPK) that occurred at 10‐ to 100‐fold lower concentrations, and that were linked to a phosphoinositide 3‐kinase pathway. These results suggest that MIP‐3α can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP‐3α in eosinophils is a significant signaling pathway for migration induction. J. Leukoc. Biol. 66: 674–682; 1999.

A small molecule CRTH2 antagonist inhibits FITC-induced allergic cutaneous inflammation

A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.

Antagonism of CRTH2 ameliorates chronic epicutaneous sensitization-induced inflammation by multiple mechanisms

Prostaglandin D(2) (PGD(2)) and its receptor chemoattractant receptor homologous molecule expressed on T(h)2 cells (CRTH2) have been implicated in the pathogenesis of numerous allergic diseases. We investigated the role of PGD(2) and CRTH2 in allergic cutaneous inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated cutaneous inflammation caused by either repeated epicutaneous ovalbumin or FITC sensitization. Gene expression and ELISA analysis revealed that there was reduced pro-inflammatory cytokine mRNA or protein produced. Importantly, the CRTH2 antagonist reduced total IgE, as well as antigen-specific IgE, IgG1 and IgG2a antibody levels. This reduction in antibody production correlated to reduced cytokines produced by splenocytes following in vitro antigen challenge. An examination of skin CD11c(+) dendritic cells (DC) showed that in mice treated with the CRTH2 antagonist, there was a decrease in the number of these cells that migrated to the draining lymph nodes in response to FITC application to the skin. Additionally, naive CD4(+) T lymphocytes co-cultured with skin-derived DC from CRTH2 antagonist-treated mice showed a reduced ability to produce a number of cytokines compared with DC from vehicle-treated mice. Collectively, these findings suggest that CRTH2 has a pivotal role in mediating the inflammation and the underlying immune response following epicutaneous sensitization.

T cell recognition motifs of an immunodominant peptide of myelin basic protein in patients with multiple sclerosis: structural requirements and clinical implications

Myelin basic protein (MBP)‐reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83 – 99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2‐ and DR4‐restricted T cell clones specific for the MBP83 – 99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine‐substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83 – 99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2‐ and DR4‐restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81 – 95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83 – 99‐specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide‐based therapy for MS.

Murine bone marrow-derived mast cells express chemoattractant receptor-homologous molecule expressed on T-helper class 2 cells (CRTh2)

Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D(2). We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD(2) receptor; chemoattractant receptor-homologous molecule expressed on T(h) class 2 cells (CRT(h)2). Activation of CRT(h)2 on BMMC by PGD(2) or the CRT(h)2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D(2) (DK-PGD(2)), resulted in signaling response including Ca(2+) mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT(h)2, Compound A. Activation of CRT(h)2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD(2) and DK-PGD(2) induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD(2) injection. Both these processes were inhibited by the CRT(h)2 antagonist. These results raise the possibility that the functional consequences of the PGD(2)-CRT(h)2 interaction on mast cells may be relevant in allergic inflammation.