High MAPK p38 activity and low level of IL-10 in intermittent claudication as opposed to stable angina.

AIM: The aim of the present pilot study was to relate the activity of MAPK p38 with the levels of pro- and anti-inflammatory cytokines in a small cohort of patients with either stable angina (N=5) or intermittent claudication (N=5) compared to healthy controls (N=10). METHODS: The activity of MAPK p38 was determined in peripheral blood mononuclear cells, isolated from whole blood by western blot using phospho-specific anti-MAPK p38 antibodies. Cytokine levels of 11 pro- and anti-inflammatory cytokines were determined from the serum using flow cytometry. RESULTS: We found a significant elevation of the MAPK p38 activity in the intermittent claudication group (P=0.0027) compared with the healthy control group whereas the stable angina group showed similar MAPK p38 activity as the healthy control group. The IL-10 level in serum found in the stable angina group was significantly higher compared with both the healthy control group (P=0.0116) and the intermittent claudication group (P=0.0317). CONCLUSION: Our results imply that there is a casual relationship between increased levels of the anti-inflammatory cytokines IL-10 and IL-4 and the activity of the MAPK p38. Possibly has IL-10 a protective role that down-regulates the activity of MAPK p38 and thereby further inflammatory processes in stable angina patients.

Influence on spontaneous tissue inflammation by the major histocompatibility complex region in the nonobese diabetic mouse.

We investigated the role of the major histocompatibility complex (MHC) region in the specificity of autoimmunity by analysing specifically the development of sialadenitis, but also insulitis, nephritis and autoantibody production in autoimmune-prone nonobese diabetic (NOD) mice where the MHC H2g7 haplotype had been exchanged for the H2q (NOD.Q) or H2p (NOD.P) haplotype. The exchange of H2 haplotype did not affect the frequency of sialadenitis because the H2q and H2p congenic NOD strains developed sialadenitis with the same incidence as NOD. However, the severity of sialadenitis varied among the strains, as NOD.Q >NOD >NOD.P. At 11-13 weeks of age, the NOD.Q (H2q) female mice developed more severe sialadenitis compared to NOD.P (H2p) (P=0.038). At 20 weeks, the NOD (H2g7) female mice showed more severe sialadenitis than NOD.P (P=0.049). This is in contrast to the development of insulitis in the present strains, because the incidence of insulitis was almost completely inhibited by the replacement of the H2g7 haplotype of NOD. The incidence of insulitis in NOD.Q was 11-22%, compared to 75% in NOD, which correlated well with lower titres of anti-glutamic acid decarboxylase (anti-GAD) antibodies in NOD.Q compared to NOD (P=0.009). However, the introduction of the H2q haplotype into the NOD strain instead directed the autoimmune response towards the production of lupus types of autoantibodies, because the incidence of antinuclear antibodies (ANA) in NOD.Q was 89% compared with 11% in NOD.P and 12% in NOD mice, which in turn correlated with a high incidence of nephritis in NOD.Q compared to NOD. Consequently, we show that different haplotypes of MHC are instrumental in directing the specificity of the spontaneous autoimmune inflammation.

The H2-Ab gene influences the severity of experimental allergic encephalomyelitis induced by proteolipoprotein peptide 103-116.

Immunization of H2(p) and H2(q) congenic C3H mouse strains with the PLP 103-116 peptide elicited two distinct experimental allergic encephalomyelitis (EAE) disease courses. C3H.Q (H2(q)) mice developed an acute-phase disease with classical ascending paralytic signs whereas C3H.NB (H2(p)) developed a highly variable disease course with symptoms originating from CNS above the spinal chord. C3H.Q lacks functional H2-E molecules and share H2-Aalpha with C3H.NB. To examine if the differences found at positions 85, 86, 88, and 89 in the Abeta-chains account for disease susceptibility, H2(q) mice were made transgenic with the Ab(p) gene. The Ab(p)-transgenic mice on the C3H.Q background developed a more severe disease course, demonstrating the importance of class II. However, the onset was not affected and the disease showed a classical ascending paralysis similar to the C3H.Q suggesting that the observed brain symptoms were related to nonclass II genes. Inhibition studies performed on affinity purified MHC class II molecules indicated that the PLP 103-116 peptide bound to A(p) with slightly higher affinity than to A(q). Both A(q) and A(p) formed long-lived stable complexes (t(1/2)>24 h) with the PLP 103-116 peptide, but a higher amount of the peptide was loaded on to A(p) compared with A(q). An F2 gene segregation experiment, in which the low PLP 103-116 binding A(r) molecule and the high binding A(p) molecule could be compared for the influence on the disease susceptibility, indicated a role for both peptide binding affinity and non-MHC genes. Based on our results, we conclude that the H2-Ab gene controls severity of EAE but not necessarily the onset or type of disease course and that affinity of the disease-promoting peptide for the class II molecule is a critical pathogenic factor.

Genetic control of collagen-induced arthritis in a cross with NOD and C57BL/10 mice is dependent on gene regions encoding complement factor 5 and FcgammaRIIb and is not associated with loci controlling diabetes.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune-mediated diseases such as diabetes and Sjögren's syndrome. To investigate whether NOD genes also promote autoimmune-mediated arthritis we established a NOD strain with an MHC class II fragment containing the A(q) class II gene predisposing for collagen induced arthritis (NOD.Q). However, this mouse was resistant to arthritis in contrast to other A(q) expressing strains such as B10.Q and DBA/1. To determine the major resistance factor/s, a genetic analysis was performed. (NOD.Q x B10.Q)F1 mice were resistant, whereas 27% of the (NOD.Q x B10.Q)F2 mice developed severe arthritis. Genetic mapping of 353 F2 mice revealed two loci associated with arthritis. One locus was found on chromosome 2 (LOD score 9.8), at the location of the complement factor 5 (C5) gene. The susceptibility allele was from B10.Q, which contains a productive C5 encoding gene in contrast to NOD.Q. The other significant locus was found on chromosome 1 (LOD score 5.6) close to the Fc-gamma receptor IIb gene, where NOD carried the susceptible allele. An interaction between the two loci was observed, indicating that they operate on the same or on interacting pathways. The genetic control of arthritis is unique in comparison to diabetes, since none of these loci have been identified in analysis of diabetes susceptibility.

Screening of several H-2 congenic mouse strains identified H-2(q) mice as highly susceptible to MOG-induced EAE with minimal adjuvant requirement.

We identified H-2(q) as a susceptible genotype for MOG-induced EAE by systematic screening of a series of H-2 congenic B10 mouse strains. A series of H-2(q)-bearing strains with divergent gene backgrounds were subsequently investigated. DBA/1 mice were highly susceptible to MOG(1-125)- and MOG(79-96)-induced EAE in the absence of pertussis toxin. Immunisation with MOG(1-125) and MOG(79-96) induced an autoreactive T-cell response in DBA/1 mice. Brain histopathology revealed T-cell and macrophage-infiltrated lesions with associated demyelination. The important features which make this an appropriate model of human disease are high sensitivity to MOG and dependence of an immunodominant peptide region homologous to that implicated in multiple sclerosis.

Allelic variations in rat MHC class II binding of myelin basic protein peptides correlate with encephalitogenicity.

The impact of the strength and promiscuity of the self peptide-MHC class II interaction on susceptibility to autoimmune disease is uncertain. Here we studied allelic differences in the affinity of rat MHC class II molecules for myelin basic protein (MBP) peptides spanning from position 63 to 106. Predominantly peptides from this region are immunogenic in the rat and the MHC class II region determines if the response is disease promoting or disease protective. Strikingly, RT1.B (DQ-like) molecules showed much more allelic variation of MBP peptide binding than RT1.D (DR-like) molecules. Moderate to strong binding of particular MBP peptides correlated with their previously documented encephalitogenicity. Moreover, the differences in disease susceptibility to certain MBP peptides observed in the different rat strains were clearly reflected in the allelic diversity of the peptide binding profiles. In conclusion our findings demonstrate that disease-inducing stretches of MBP generally comprise good binding peptides.

Type II collagen in cartilage evokes peptide-specific tolerance and skews the immune response.

T cell recognition of type II collagen (CII) is a crucial event in the induction of collagen-induced arthritis in the mouse. Several CII peptides have been shown to be of importance, dependent on which MHC haplotype the mouse carries. By sequencing the rat CII and comparing the sequence with mouse, human, bovine and chicken CII, we have found that the immunodominant peptides all differ at critical positions compared with the autologous mouse sequence. Transgenic expression of the immunodominant Aq-restricted heterologous CII 256-270 epitope inserted into type I collagen (TSC mice) or type II collagen (MMC-1 mice) led to epitope-specific tolerance. Immunization of TSC mice with chick CII led to arthritis and immune responses, dependent on the subdominant, Aq-restricted and chick-specific CII 190-200 epitope. Immunization of F1 mice, expressing both H-2q and H-2r as well as transgenic expression of the Aq-restricted CII 256-270 epitope in cartilage, with bovine CII, led to arthritis, dependent on the Ar-restricted, bovine-specific epitope CII 607-621. These data show that the immunodominance of CII recognition is directed towards heterologous determinants, and that T cells directed towards the corresponding autologous epitopes are tolerated without evidence of active suppression.

Genetic influence on disease course and cytokine response in relapsing experimental allergic encephalomyelitis.

A protracted and relapsing form of experimental allergic encephalomyelitis (EAE) develops in the DA rat after immunization with rat spinal cord homogenate (SCH) emulsified in incomplete Freund's adjuvant (IFA). The genetic influence on this model has been analyzed by immunizing MHC congenic strains on both LEW and DA genetic backgrounds, and recombinant inbred strains between DA and E3 rats. An in situ hybridization assay was used to examine the expression of mRNA for IFN-gamma, IL-4, IL-10 and transforming growth factor (TGF)-beta both in sections of spinal cords and the antigen-induced expression for these cytokines by splenocytes after in vitro stimulation with encephalitogenic MBP peptides. The susceptibility of relapsing EAE after immunization with SCH in IFA in the DA strain, but not the E3 strain, was correlated with a lack of expression for TGF-beta in the spinal cord. The recombinant inbred DXEB rats developed a severe EAE while surprisingly no signs of disease were observed in the DXEA strain, which shares the MHC region with the DXEB strain, after immunization with the MBP 63-87 peptide. Resistance to relapsing EAE in the DXEA strain correlated with increased non-MHC controlled expression for TGF-beta and lack of IFN-gamma in the spinal cord. The same pattern of cytokine expression was seen in splenocytes after stimulation in vitro with the MBP 63-87 peptide. A spreading of the immune response to the MBP 87-110 peptide was seen. Non-MHC genes controlled the quality of this response: splenocytes from MBP 63-87 immunized DXEB rats responded in vitro towards the MBP 87-110 peptide by expressing mRNA for IFN-gamma, IL-10 and IL-4, whereas in the DXEA strain the corresponding response involved IL-4 and TGF-beta. Taken together these data show that non-MHC controlled expression of mRNA for TGF-beta is associated with resistance to EAE.

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules.

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

Novel galanin receptor ligands.

Galanin is a neuroendocrine peptide which is 29/30 amino acids in length and is recognised by G-protein-coupled central nervous system receptors via its N-terminus. We synthesised several galanin receptor ligands and fragments around C-terminal extensions of galanin(1-13) to yield chimeric peptides with C-terminals corresponding to bioactive peptides like bradykinin(2-9), mastoparan, neuropeptide Y(25-36) or substance P(5-11), respectively. We also synthesised short galanin analogs in which galanin(1-13) was C-terminally elongated with Lys14; different pharmacologically active small molecules were then attached to the epsilon-amino group of Lys14. Several cysteine-substituted linear and ring closed analogs of galanin(1-9) and galanin(1-16) were also synthesised. The equilibrium binding constants for these peptides at hypothalamic galanin receptors were determined and found in the subnanomolar to micromolar range. The large number of peptides and their binding affinities presented here permit structure-activity relationship analysis of peptide-type ligands to galanin receptors.

Major histocompatibility complex-controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific.

The myelin basic protein (MBP) peptide 63-88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89-101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89-101 as compared to MBP 63-88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87-110) induced EAE associated with the same MHC haplotypes as the 89-101 peptide, except in LEW.1N (RT1 pi) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-beta responses to the peptide in vitro. In vivo depletion of CD8+ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87-110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.

Expression of Bruton's agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.