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.

MIP-3alpha induces human eosinophil migration and activation of the mitogen-activated protein kinases (p42/p44 MAPK).

The CC chemokine macrophage inflammatory protein-3alpha (MIP-3alpha) 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-3alpha. Messenger RNA for the MIP-3alpha 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-3alpha-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-3alpha can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP-3alpha in eosinophils is a significant signaling pathway for migration induction.

Activation of mitogen-activated protein kinase regulates eotaxin-induced eosinophil migration.

Eotaxin is a potent eosinophil chemoattractant that plays an important role in regulating eosinophil tissue levels both in healthy individuals and in diseases associated with significant eosinophil infiltrates, such as the allergic inflammation observed in asthma. Here, we demonstrate that treatment of eosinophils with eotaxin induces the phosphorylation of the mitogen-activated protein kinases (MAPKs) p42 and p44, leading to kinase activation. Blockade of MAPK activation by the MAPK kinase inhibitor PD98059 leads to a dramatic decrease in eotaxin-induced eosinophil rolling in vivo and chemotaxis in vitro. This blockade in the leukocyte migration process is consistent with the observed inhibition of actin polymerization and rearrangement within the eosinophil following treatment with MAPK inhibitor. It is suggested, therefore, that the intrinsic mechanism of eotaxin-induced eosinophil rolling and migration involves activation of the p42/p44 MAPK, possibly through regulation of the cytoskeletal rearrangements necessary for chemotaxis.

Human thymocytes express CCR-3 and are activated by eotaxin.

Eotaxin has been characterized as a chemokine involved in eosinophil activation; however, mRNA for this C-C chemokine has been shown to be constitutively expressed in thymus. Immunohistochemical analysis showed a punctate distribution pattern, with eotaxin expression localized mainly in the medulla and in Hassle's corpuscles. Moreover, the receptor for eotaxin, CCR-3, was detected on thymocytes, with the highest level of expression being on the CD8 single-positive population. Equilibrium binding analyses on unfractionated thymocytes demonstrated specific 125I-eotaxin binding profiles comparable with CCR-3 transfectants. Eotaxin induced cell migration and mobilization of intracellular calcium in all thymocytes except the immature CD4(-)/CD8(-) population. Eotaxin also induced the secretion of the chemokines interleukin-8, RANTES, and macrophage inflammatory protein-1beta from thymocyte cultures in vitro. These results suggest that eotaxin-induced thymocyte activation may have important physiological implications for lymphocyte mobilization within and from this lymphoid organ.

Differential signaling and hierarchical response thresholds induced by an immunodominant peptide of myelin basic protein and an altered peptide ligand in human T cells.

Changes in peptide antigen concentration or structure can have a profound effect on T cell responsiveness by inducing selected T cell effector functions. In this study, we have compared the biological responses of an MBP83-99-specific human Th0 T cell clone (TCC) stimulated with increasing concentrations of native peptide or an altered peptide ligand (APL). Our results show that the hierarchy of response thresholds for proliferation and cytokine secretion is similar for native peptide and APL. However, because a much higher concentration of the APL is required to evoke the same degree of response, the cytokine profile is shifted towards a Th2-like response relative to the same concentration of native peptide. In addition, we observed qualitative differences in TCR signal transduction triggered by native peptide and a weak agonist APL even at concentrations that elicit similar biological responses. Thus, the relationship between TCR signaling and biological responses may be more complex than previously recognized.

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.

Macrophage inflammatory protein-1beta induces migration and activation of human thymocytes.

The CC chemokine macrophage inflammatory protein 1beta (MIP-1beta), has been shown to be a chemoattractant preferentially activating CD4(+) CD45RA+ T lymphocytes. Further analysis of chemokine action on lymphocytic cells has shown the potent migration-promoting capacity of MIP-1beta on human thymocytes. The responding cells were the CD4(+) and CD8(+) single-positive (SP), as well as the CD4(+) CD8(+) double-positive (DP) populations, with little if any migratory activity on the double-negative (DN) population. The activation of thymocytes by MIP-1beta appeared to be a direct, receptor-mediated event as evidenced by the rapid mobilization of intracellular calcium, increase in proteins phosphorylated on tyrosine, and activation of the mitogen-activated protein kinase (MAPK) pathway. Radioligand binding analyses showed specific and displaceable binding of MIP-1beta to thymocytes with a Kd of approximately 1 nmol/L, a profile that was comparable with MIP-1beta binding to CCR-5-transfected NIH 3T3 cells. In addition, CCR-5 mRNA was detected in total thymocyte populations indicating that activation of thymocytes by MIP-1beta may occur through binding to CCR-5. Further dissection of the subpopulations showed that only the DP and CD8(+) SP populations expressed CCR-5 and expression data on these two populations was confirmed using anti-CCR-5 monoclonal antibody. These data may be suggestive of a role for MIP-1beta in human thymocyte activation, and show a potential route for HIV infectivity in the developing immune system.

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.

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.

Immunosuppressive phenotype of corticotropin-releasing factor transgenic mice is reversed by adrenalectomy.

Stress elicits a wide range of physiological changes involving the nervous, endocrine, and immune systems. Corticotropin-releasing factor (CRF) plays a key role in orchestrating this response, activating both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, resulting in release of corticosteroids. The present study examines the immunological phenotype and responsiveness of CRF-transgenic (CRF-Tg) mice. The immune system of the CRF-Tg animals has profound changes compared to littermate controls, including a marked reduction in both cell number and immune responsiveness. There were also phenotypic changes in the lymphocytic composition of the various lymphoid organs, most notably in the spleen, where CRF-Tg mice had a greater percentage of T lymphocytes compared to littermate controls. Adrenalectomy of CRF-Tg reversed the immunological phenotype observed and restored immune responsiveness. These results demonstrate that CRF overexpression leads to profound impairment on lymphocyte development and function mediated via corticosteroids.

Development and characterization of v-myc/v-raf-transformed murine fetal thymocyte cell lines.

Transformed murine fetal thymocyte cell lines were derived by incubating fetal thymic organ cultures with a v-myc/v-raf-containing retroviral construct in order to model developmental stages within the early triple negative (CD3-CD4-CD8-) thymocyte population. The resulting 10 cell lines had a lymphoid morphology, were all CD44+, CD90+, and were triple negative by surface antigen analysis. The cell lines, however, were distinguishable by differences in the expression of T cell-associated and T cell-specific genes. The CD3 genes were observed to be discoordinately expressed, in that CD3 gamma chain gene expression was detected in 2 cell lines in the absence of CD3 delta and epsilon expression. Expression of the CD3 gamma chain gene was observed in cell lines without the expression of other T cell-specific genes or T cell receptor rearrangement and may be one of the earliest T cell-specific genes to be expressed. The transcription factor Ikaros was expressed in all 10 cell lines, whereas the transcription factor TCF1 alpha was expressed only in the 2 most differentiated lines. In 8 cell lines, expression of partial TCR beta and/or TCR alpha transcripts was observed by Northern blot. In several lines, expression of rearranged TCR alpha transcripts in the absence of TCR beta transcripts was demonstrated; however, TCR beta DJ rearrangements were observed by Southern blot in all but 1 of these cell lines. Thus, these cell lines, ordered based on the general pattern of additive gene expression observed, may reflect various stages of triple-negative thymocyte differentiation and provide an in vitro mechanism to elucidate some of the molecular events involved in early thymocyte development.

TCR-mediated death of mature T lymphocytes occurs in the absence of p53.

The p53 protein plays an important role in various forms of thymocyte apoptosis, however its role in mature T lymphocyte death caused by TCR stimulation has not been examined. We demonstrate here that T cell blasts derived from mice containing a germ-line deficiency of the p53 tumor suppressor gene are susceptible to TCR-induced apoptosis to the same degree as wild-type T cells. TCR stimulation of both resting and proliferatingT cells results in up-regulated expression of the p53-induced genes Bax and p21, and the induction of these genes appears reduced in T cells that are deficient in p53. Thus, while activation of p53-dependent genes following TCR stimulation is defective in p53-/- T cells, there is no impairment in their ability to undergo apoptosis. These results suggest that TCR-mediated apoptosis of mature T cells takes place via a pathway that is independent of p53.

Analysis of the CD4 coreceptor and activation-induced costimulatory molecules in antigen-mediated mature T lymphocyte death.

We have compared the signaling requirements for activation and lymphokine production in mature T lymphocytes to those required for TCR-driven programmed cell death (PCD). Both processes require TCR engagement and ligation of the CD4 coreceptor in the case of a T cell clone that recognizes Ag in the context of an MHC class II molecule. By contrast, stimulation through the CD28/B7 pathway does not appear to positively or negatively influence TCR-induced PCD, although it was required for IL-2 production in both resting and proliferating T cells. T cells that had been activated and induced to proliferate with IL-2 were found to express high levels of IL-2 mRNA upon TCR rechallenge, without a requirement for accessory cells. This was due to a strong up-regulation of the B7-1 molecule, but not the B7-2 molecule, on the T cell surface. These T cells that strongly costimulate each other are highly susceptible to TCR-induced death providing independent evidence that costimulatory signals are not protective. Thus, these results provide evidence that in mature T cells there exists a difference in the requirement for CD28 to achieve activation and IL-2 production compared with TCR-mediated PCD.

Two mechanisms of antigen-specific apoptosis of myelin basic protein (MBP)-specific T lymphocytes derived from multiple sclerosis patients and normal individuals.

Several stimuli induce mature T cells to undergo apoptosis or programmed cell death (PCD) including specific Ag. We have demonstrated previously that Ag induces the death of encephalitogenic T cells in vitro and deletion in vivo, leading to amelioration of autoimmune encephalomyelitis. We have now examined whether activated, myelin basic protein (MBP)-specific human T cells may be eliminated by Ag-induced PCD. We demonstrate that activated MBP-specific T cell lines (TCL) undergo the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when given a TCR challenge. We found evidence that two mechanisms led to apoptosis: a propriocidal mechanism that was highly Ag-specific and dependent on the dose of exogenously added rIL-2, and a cytolytic mechanism in which MBP-specific TCL lysed B cell targets and engaged in considerable "fratricidal" cytolysis of other MBP-specific T cells. These two pathways leading to MBP-specific apoptotic death could be distinguished by their glucocorticoid sensitivity. Glucocorticoid treatment significantly blocked MBP-induced propriocidal apoptosis but had no effect on T cell cytolysis of B cell targets. Although it has been proposed that autoimmune disease could result from the failure of normal deletional mechanisms, this preliminary survey of MBP-reactive mature TCL from multiple sclerosis patients revealed that such cells are highly susceptible to TCR-induced PCD and comparable with TCL from normal subjects. Thus, therapeutic strategies based on Ag-induced PCD of T lymphocytes may be feasible in man.

Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells.

We have shown that T cells vigorously cycling in response to growth lymphokines are driven into apoptosis by potent TCR restimulation. This process, termed propriocidal regulation, appears to be a normal feedback inhibitory mechanism to prevent excessive T cell proliferation and lymphokine production. Exposure of T cells to repeated high dose antigen treatments creates the conditions just described by activating T cells, and stimulating the production of growth lymphokines and their receptors. High growth lymphokine levels induced by the large amount of antigen present, stimulate vigorous cycling. The continued presence of high antigen levels subjects the cycling T cells to strong TCR restimulation as they enter the vulnerable S phase, inducing apoptosis in T cells responsive to the administered antigen. Thus, simple, repetitive, intravenous administration of high dose antigen may be used to delete potentially destructive clones of T cells, resulting in a state of peripheral tolerance. This has obvious therapeutic potential in disorders where the elimination of pathogenic T cell clones could be beneficial. We have described in EAE, an animal model for MS, that high dose MBP therapy is effective in preventing CNS pathology and the onset of disease as well as reducing the severity of the clinical symptoms of established EAE. We are currently involved in expanding this approach to other animal models of autoimmunity and graft rejection, as well as refining the immunotherapy in the EAE model with the objective of developing a clinical therapy for human demyelinating disease.

Ligand-induced apoptosis of mature T lymphocytes (propriocidal regulation) occurs at distinct stages of the cell cycle.

We previously demonstrated that mature T lymphocytes responding to either IL-2 or IL-4 undergo apoptosis upon T cell antigen receptor stimulation, and have termed this potential negative feedback pathway propriocidal regulation. Using cell cycle inhibitors, we now show that T cell growth lymphokines cause the entry of T cells into vulnerable stages of the cell cycle in which T cell receptor occupancy causes apoptosis.

Propriocidal apoptosis of mature T lymphocytes occurs at S phase of the cell cycle.

We found that mature nontransformed CD4+ and CD8+ T lymphocytes could be made susceptible to T cell receptor(TcR)-mediated apoptosis by pretreatment with interleukin-4 (IL-4) or interleukin-2 (IL-2). The degree of susceptibility to death could be correlated with the level of cell cycling as measured by thymidine incorporation, cell doubling times, or the number of cells incorporating bromodeoxyuridine during S phase. However, using pharmacologic cell cycle blocking agents, we found that progression through the cell cycle was not required for cell death. Rather, we found that cells must be in a certain phase of the cell cycle to be susceptible to TcR-mediated death. Cells blocked in G1 phase were resistant to T cell receptor-induced apoptosis, whereas cells blocked in S phase were susceptible. These observations suggest that an important feature of growth lymphokines is their ability to drive T cells into portions of the cell cycle where they are sensitive to antigen receptor-induced apoptosis. Furthermore, these results provide additional evidence that the T cell growth lymphokines IL-2 and IL-4 may participate in the down-regulation of T cell responses by apoptosis-a pathway we have termed "propriocidal regulation".

Amplification of major histocompatibility complex class II gene diversity by intraexonic recombination.

The roles of mutational and recombinational processes in the diversification of the exon encoding the antigen binding site in the murine major histocompatibility complex class II gene Ab were assessed by phylogenetic analysis of allelic nucleotide sequences. A total of 46 alleles of Ab exon 2 from 12 Mus species or subspecies and 2 Rattus species were sequenced after amplification by the polymerase chain reaction. Reliable allelic genealogies could not be determined by phylogenetic analyses, due to extensive homoplasy in the data set. This homoplasy results from the shuffling of polymorphisms between alleles by recombinational processes, indicating that polymorphisms in the antigen binding site encoded by Ab are generated by a combination of two processes. First, the accumulation of point mutations has produced highly divergent polymorphic sequence motifs in five regions of Ab exon 2, each encoding a portion of the binding site. Some of these motifs have persisted as polymorphisms in rodents since before the divergence of mouse and rat (greater than 10 million years ago). The second process mediating Ab diversification involves the shuffling of these polymorphic sequence motifs into numerous allelic combinations by repeated intraexonic recombination. Site-specific hyperrecombinational mechanisms are not involved in this process within the exon. We postulate that these mechanisms continuously generate new Ab alleles with highly divergent binding sites from which alleles with advantageous antigen-binding properties are selectively maintained by some form of balancing selection.