Mapping of a second recombination hot spot within the I-E region of the mouse H-2 gene complex.

The crossover points of nine intra-I region recombinant mouse strains were determined by restriction fragment analysis. The recombinants were examined for the presence of k and p haplotype specific DNA restriction endonuclease sites. These restriction sites were a Sac I site between the E beta and E beta 2 genes, a Hpa I site within the E beta 2 gene, and a Rsa I site approximately 1 kb to the right of the E alpha gene. Seven of the recombinants were found to have crossovers between the Hpa I and the Rsa I site. This analysis suggests that a recombination hot spot exists within this segment. This segment is approximately 12-14 kb, and contains the E alpha gene and the intervening sequence between the E beta 2 and E alpha genes.

Serological and biochemical identification of hybrid Ia antigens.

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules by serological and biochemical studies. Lipopolysaccharide-stimulated splenic lymphocytes from (B10 X B10.D2)F1 expressed Ia.22 although both the parents were negative. Similarly [D2.GD X B10.A(5R)]F1 cells expressed Ia.23, whereas D2.GD and B10.A(5R) lacked it. Ia.22 can be generated by gene complementation of Ak-Ek, Ab-Ed, Ab-Ek, As-Ed, and As-Ek, whereas Ia.23 can be generated by Ad-Ed, Ad-Ek, and Ad-Ep. Other possible complementing combinations are under study. The role of Ia.22 and 23 in mixed lymphocyte reactions and immune responses are discussed.

Molecular biology of murine MHC class II genes.

The murine class II genes are contained in the I region of the major histocompatibility complex (MHC). In the mouse, there are seven class II genes: A beta 3, A beta 2, A beta, E beta, E beta 2, A alpha, and E alpha. The A beta and A alpha genes code for the two polypeptide chains that form the I-A immune response molecule. The E beta and E alpha genes code for the two polypeptide chains that form the I-E immune response molecule. This review covers the genetic organization of the I region and the exon-intron structure of the class II genes. This review also discusses site-directed mutagenesis and exon shuffling studies and the effect of these changes on the function of Ia genes. Regulation of the cellular expression of Ia genes is discussed with emphasis on recent studies involving class II transgenic mice. Also, studies mapping recombination hotspots within the E alpha and E beta genes are reviewed.

Psychological stress-induced modulation of interleukin 2 receptor gene expression and interleukin 2 production in peripheral blood leukocytes.

We explored the expression of the interleukin 2 receptor (IL-2R) and the synthesis of IL-2R messenger RNA by peripheral blood leukocytes obtained from medical students experiencing examination stress in three independent studies. The peripheral blood leukocytes obtained at low-stress baseline periods had significantly higher percentages of IL-2R-positive cells when compared with cells obtained from the same individuals during examinations. In addition, IL2-R messenger RNA in peripheral blood leukocytes decreased significantly during examination periods in a subset of 13 subjects. In one study, we found an increase in the accumulation of interleukin 2 in cultures of cells showing down regulation of IL-2R expression and IL-2R messenger RNA levels. While there are ample data demonstrating stress-associated decrements in the immune response in humans and animals, these data provide the first evidence that this interaction may be observed at the level of gene expression. The data suggest one mechanism whereby the central nervous system modulates the immune response during psychological stress.

Iron transport into mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage cell line.

Nramp1 is an important determinant of innate resistance of macrophages to the growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobacterium avium-infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1-mediated iron transport into phagosomes. Treatment of Nramp1(Gly169) macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly. We found that macrophage-activating cytokines, including TNF-alpha, IFN-gamma, IL-1alpha, and GM-CSF, when added prior to M. avium, increased the transport of iron into the phagosome. This increase in iron transport was not a result of an increased amount of Nramp1 protein in the phagosome nor to new protein synthesis. Treatment of Nramp1(Gly169)-transfected macrophages with inhibitors of protein kinase C (PKC) diminished the import of iron into the phagosomes. Iron import was inhibited by an anti-Nramp1 antibody against the putative fourth outer-loop region of Nramp1 but not by an anti-Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 in the phagosome membrane and on the transport of iron is discussed.

IFN-gamma increases cathepsin H mRNA levels in mouse macrophages.

Expression of major histocompatibility complex (MHC) class II molecules and ability to present antigen to T lymphocytes is acquired upon activation of the macrophage by interferon-gamma (IFN-gamma). Little information is available concerning immune regulation of protease gene expression in mouse macrophages. We have isolated a cDNA clone for cathepsin H, a lysosomal cysteine proteinase from a cDNA subtraction library of mouse macrophage genes induced by IFN-gamma, and have characterized its expression. The level of cathepsin H mRNA increased in mouse peritoneal macrophages following addition of IFN-gamma. Cathepsin H mRNA levels began to increase 8 h after the addition of IFN-gamma and was maximal at 24-48 h. This increase was concordant in time with appearance of MHC class II E beta mRNA and Ia invariant chain mRNA. The increase in cathepsin H mRNA levels by IFN-gamma was dose dependent. Cycloheximide treatment of peritoneal macrophages inhibited the increase in cathepsin H mRNA levels induced by IFN-gamma, suggesting that the increase in cathepsin mRNA levels requires de novo protein synthesis. Lipopolysaccharide and cytokines interleukin-2 (IL-2), IL-4, IL-10, and tumor necrosis factor alpha were found to have no effect on cathepsin H mRNA levels in mouse peritoneal macrophages.

Cloning and characterization of a novel cDNA that is IFN-gamma-induced in mouse peritoneal macrophages and encodes a putative GTP-binding protein.

Macrophage activation by IFN-gamma results in a cascade of gene expression. To identify genes activated in mouse peritoneal macrophages by IFN-gamma, we created a cDNA subtraction library of IFN-gamma-induced genes. We have isolated from this subtraction library a novel cDNA clone, called Mg21, whose mRNA is absent in unstimulated mouse peritoneal macrophages and is induced to high levels within 4 h following the addition of IFN-gamma. Induction of Mg21 mRNA by IFN-gamma occurred in the presence of cycloheximide, indicating that expression of Mg21 mRNA does not require protein synthesis. A small amount of Mg21 mRNA was also induced by LPS, but not by IL-2, IL-4, IL-10, or TNF-alpha. The DNA sequence of Mg21 is 1617 nucleotides and contains an open reading frame that codes for a protein of 415 amino acids with a predicted molecular weight of 47,106 Da. The predicted amino acid sequence lacks a signal sequence or transmembrane segments, indicating that the protein is an intracellular protein. Computer search of the GenBank and EMBL databases indicates that this cDNA clone is unique but has 57% sequence identity with IRG-47, which is a mouse gene induced by IFN-gamma in pre-B and B lymphocyte cell lines. IRG-47 encodes an intracellular protein that contains three conserved protein motifs present in GTP-binding proteins. Analysis of the protein sequence of Mg21 showed that these three conserved protein motifs are also present in Mg21.

Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169.

The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1ASp169 (susceptible) alleles was assessed. We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells. In contrast, the rate of Fe import by latex-bead phagosomes isolated from resistant cells was more than double the rate by latex-bead phagosomes from susceptible cells. Similarly, phagosomes isolated from resistant cells that had been pre-labeled with 55Fe-citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells. Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1cGly169-transfected macrophages but not by macrophages transfected with the susceptible allele. These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium-containing phagosome where it serves as a catalyst for the Haber-Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth.

Partial suppression of Theiler's virus-induced demyelination in vivo by administration of monoclonal antibodies to immune-response gene products (Ia antigens).

In vivo administration of monoclonal antibody reactive with major histocompatibility complex-encoded Ia molecules (I-As) partially suppressed inflammation and demyelination in the spinal cord of SJL/J (H-2s) mice persistently infected with Theiler's murine encephalomyelitis virus. Demyelination was decreased if antibody was given at the time of virus inoculation or after inflammation had been established in the spinal cord. The decrease in demyelination was independent of isolation of infectious virus from the CNS or of serum titers of immunoglobulin to purified viral antigen. Thus, Theiler's virus-induced demyelination is mediated, in part, by immune cells that carry Ia class II molecules.

Cross-reactions of anti-I-Ek monoclonal antibodies with subsets of I-Ab molecules.

Monoclonal antibodies H9-15.4 and H39-459 were derived from an A.TH anti-A.TL immunization. Antibodies H9-15.4 and H39-459 were found to be directed against the I-Ek molecule with positive reactions against B10.A (AkEk) and B10.S(9R) (AsEk) but not B10.A(4R) (AkEb). The monoclonal antibodies were also found to react with the b and q haplotypes, which do not express an I-E molecule. Sequential precipitation analysis showed that in the b haplotype, H9-15.4 and H39-459 react with the I-Ab molecule. These results showed that H9-15.4 and H39-459 recognize determinants shared by I-Ek and I-Ab molecules, suggesting that I-A and I-E molecules may have a common evolutionary origin, possibly through gene duplication. Sequential immunoprecipitation analysis of I-Ab molecules precipitated by H9-15.4 and H39-459 also suggested that these monoclonal antibodies recognize subsets of I-Ab molecules. Pretreatment with the 17-227 monoclonal anti-I-Ab antibody (Ia.15) had no effect on I-Ab molecules precipitated by H9-15.4 and H39-459. Also, pretreatment with H9-15.4 and H39-459 had no effect on I-Ab molecules immunoprecipitated by 17-227. Also H9-15.4 and H39-459 only partially cleared Ia molecules immunoprecipitated by each other. These results suggest that 17-227, H9-15.4, and H39-459 detect a minimum of four subsets of I-Ab molecules. To account for these observations, it is proposed that during evolution of the mouse H-2 complex, in addition to gene duplication of ancestral gene(s) to yield the genes encoding E alpha A alpha, A beta, and E beta polypeptide chains, further gene duplication occurred, forming multiple copies of genes encoding each Ia polypeptide chain.

Serological confirmation in Ia mutant B6.C-H-2bm12 of gene conversion.

Recently it has been suggested that a short segment of the DNA sequence of the Ia beta gene in the mutant B6.C-H-2bm12 (bm12) is derived from the Eb beta gene by a gene-conversion-like mechanism, and that the same segment is also seen in the Ek beta gene. To obtain serological evidence for this idea, we produced an antibody against the "new" determinant on the I-Abm12 molecule by immunizing A.BY mice with bm12 cells. After absorption with B6 lymphocytes to remove antibodies against background antigens, the antisera lysed bm12 cells. Typical Ia peaks were obtained by immunoprecipitation. The absorbed antiserum reacted with B10.A (Ek beta), B10.A(5R) (Eb beta) and B10.S(9R) (Es beta), but not B10. The unabsorbed antiserum is specific for Ia when tested on A background mice. The antiserum lysed spleen cells of Ik strains (A/Sn, A.AL, A.TBR1, and A.TFR1) but not Ak strain, A.TBR13 (KsAkEbSbDb), confirming the presence of antibodies against the I-Ek molecule. This anti-serum also lysed the cells from (A.BY X A.TFR5)F1, which expresses the transcomplementing Eb beta Ek alpha molecule and from the (A.SW X A.TFR5)F1 which expresses the transcomplementing Es beta Ek alpha molecule. These data are consistent with DNA sequence analyses, and show the existence of a determinant (Ia.51) generated in the bm12 mutant by a gene-conversion-like event that is also present in the I-Ek, I-Eb, and I-Es beta polypeptide chains.

Characterization of ACTH mediated suppression of MHC class II expression by murine peritoneal macrophages.

The effect of ACTH on the expression of major histocompatibility complex (MHC) class II (I-A) glycoprotein by murine peritoneal macrophages was evaluated. ACTH suppressed the expression of I-A by macrophages in a time- and dose-dependent manner. ACTH mediated its effect by decreasing the level of I-A mRNA. ACTH suppressed the expression of I-A by macrophages from mice that are susceptible to the in vivo growth of mycobacteria but did not affect the expression of I-A by macrophages from Mycobacterium bovis strain (BCG)-resistant mice. The concentrations of ACTH required to suppress I-A expression were greater than that required for an effect on adrenal steroid production and may be related to the localized production of ACTH by lymphocytes and macrophages.

Identification of I-E alpha genes in H-2 recombinant mouse strains by F1 complementation.

Nine recombinant H-2 mouse strains with crossovers in the I region between I-E-negative haplotypes f,q and I-E-positive haplotypes p,k were examined for I-E expression by microcytotoxicity dye exclusion assay. These recombinants were found to be negative for I-E expression. There are two possible genotypes in these recombinant mouse strains that could result in lack of I-E expression. Recombinants with crossovers between the E alpha gene and the S region would have both nonexpressed I-E alpha and beta genes (E beta fE alpha f, E beta qE alpha q) and recombinants with crossovers between the E beta and E alpha genes would have a nonexpressed E beta gene (E beta f or E alpha q) and a functional E alpha gene (E alpha k or E alpha p). To distinguish between these possible genotypes these recombinants were crossed to B10.A(4R), which carries a functional E alpha k gene but is I-E-negative due to a nonexpressed E alpha b gene. F1 mice were examined for transcomplementing I-E molecules by immunoprecipitation of 3H-leucine-radiolabeled detergent lysates of spleen cells with a monoclonal I-E antibody (14-4-4). Detection of a transcomplementing I-E molecule was confirmed by immunoprecipitation with a monoclonal antibody (H9-14.8) specific for the I-Ek beta polypeptide chain derived from B10.A(4R) and by tryptic peptide map comparisons. Five recombinant mouse strains were able to complement with B10.A(4R) in F1 mice to generate a transcomplementing I-E molecule, and thus have an expressed I-E alpha gene (E alpha k or E alpha p). Four recombinants did not complement with B10.A(4R) in the F1 expression of I-E molecules, and thus have nonexpressed I-E alpha genes (E alpha f or E alpha q).

Binding of alpha-adrenergic receptors stimulates the anti-mycobacterial activity of murine peritoneal macrophages.

The effects of adrenergic stimulation of the anti-mycobacterial activity of peritoneal macrophages was investigated. We found that epinephrine and norepinephrine stimulated macrophages to suppress the growth of Mycobacterium avium. Stimulation was mediated by binding to the alpha 2 adrenergic receptor. The addition of the alpha 2 agonist clonidine to cultures resulted in an inhibition of mycobacterial growth and the effect of epinephrine was blocked by the alpha-antagonist phentolamine. Treatment of the macrophages with propranolol, a beta-antagonist, potentiated the effect of epinephrine. Epinephrine mediates its effect by stimulating the expression of macrophage activation genes.

Beta2-adrenergic receptor stimulation inhibits nitric oxide generation by Mycobacterium avium infected macrophages.

Catecholamine regulation of nitric oxide (NO) production by IFNgamma-primed macrophages infected with Mycobacterium avium was investigated. Epinephrine treatment of IFNgamma-primed macrophages at the time of M. avium infection inhibited the anti-mycobacterial activity of the cells. The anti-mycobacterial activity of macrophages correlated with NO production. Using specific adrenergic receptor agonists, the abrogation of mycobacterial killing and decreased NO production by catecholamines was shown to be mediated via the beta2-adrenergic receptor. Elevation of intracellular cAMP levels mimicked the catecholamine-mediated inhibition of NO in both M. avium infected and LPS stimulated macrophages. Specific inhibitors of both adenylate cyclase and protein kinase A prevented the beta2-adrenoceptor-mediated inhibition of nitric oxide production. Beta2-adrenoreceptor stimulation at the time of M. avium infection of IFNgamma-primed macrophages also inhibited expression of iNOS mRNA. These observations show that catecholamine hormones can affect the outcome of macrophage-pathogen interactions and suggest that one result of sympathetic nervous system activation is the suppression of the capacity of macrophages to produce anti-microbial effector molecules.

Gene complementations to generate Ia antigens (Ia.23) on hybrid molecules.

Ia specificity 23 is a "combinatorial" antigen generated on a hybrid I region molecule, formed by the noncovalent binding of a 26,000- to 28,000-dalton beta polypeptide chain (Ae) coded by a gene in the I-A subregion with a 32,000- to 35,000-dalton alpha chain (E alpha) coded by a gene in the I-E subregion of the mouse H-2 gene complex. For expression of Ia.23, the Ae chain must be derived from the H-2d haplotype (I-Ad), while the E alpha can be provided by I-Ed, I-Ek, I-Ep, I-Er, I-Ev, and I-Ew3, but not I-Eb, I-Ef, I-Eq, I-Es, and I-Eu. With the exception of H-2u haplotype, all Ia.7 (I-E)-positive haplotypes can provide the permissive E alpha chain for generating Ia.23 by trans-complementation. In the H-2d haplotype, Ia.23 is generated by cis-complementation of Ad with Ed. Lymphocytes of F1 animals expressed two I-E subregion coded hybrid Ia specificities; one formed by cis-complementation and another by trans-complementation. It is postulated that such hybrid determinants are involved in the recognition and generation of immune response to antigens such as GL-Phe and cytochrome C where dual Ir gene control has been demonstrated. It is also suggested that there are two types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (these could aid in the binding between the alpha and beta chains such as Ia.7); and (2) hybrid Ia specificities which are unique interaction determinants formed by the specific association of the alpha and beta chains (e.g., Ia.22,23). These interaction gene products may be involved in antigen recognition and presentation.

Serological and biochemical analysis of Ia molecules in the I-A mutant B6.C-H-2.

Strain B6.C-H-2bm12 has a mutation in the I-A subregion of the mouse H-2 gene complex, which causes skin graft rejection, mixed lymphocyte reaction (MLR), and alterations in the expression of Ia antigens. The mutation affects the expression of Ia.3, 8, 9, 15, and 20 on normal spleen cells. When the spleen cells were stimulated with lipopolysaccharide (LPS), the expression of all Ia specificities were found except Ia.8. Ia molecules when internally labeled with 3H-leucine can be precipitated with antisera directed against Ia.3, 9, 15, and 20, but not Ia.8. When F1s are made between the mutant bm12 and unrelated haplotypes, Ia.3, 9, 15 and 20 can be detected by microcytotoxic assay on normal spleen cells, but not Ia.8. These studies suggest: (1) The mutation affects either the amount of Ia molecules expressed on normal spleen cell surfaces or the molecule is anchored improperly in the cell surface such that it is not accessible for cytotoxicity and radioiodination. (2) Specificity Ia.8, which may be a combinatorial determinant, is absent in the mutant because of a structural alteration in one of the chains, probably the beta chain. (3) The mutation does not involve the Ae chain. The significance of this finding in relation to I region-mediated allorecognition and antigen presentation is discussed.

Synergistic interaction of catecholamine hormones and Mycobacterium avium results in the induction of interleukin-10 mRNA expression by murine peritoneal macrophages.

The results of this investigation provides evidence that catecholamine hormones interact with macrophages that are infected with Mycobacterium avium resulting in the induction of IL-10 mRNA and protein. The effect of catecholamine hormones was prevented by treating the cells with the beta-adrenergic receptor antagonist propranolol but not by alpha-adrenergic antagonist phentolamine. The effect of catecholamine stimulation was mimicked by the addition of beta-2 adrenergic agonists and by the addition of cAMP to the infected macrophage cultures. These observations suggest that sympathetic nervous system activation together with microbial infection results in a synergistic interaction that could result in the control of inflammatory processes.

Stress-associated modulation of proto-oncogene expression in human peripheral blood leukocytes.

Changes in the cellular immune response associated with psychological stress were studied by using an academic stress model with medical students. The authors examined the expression of 2 proto-oncogenes, c-myc and c-myb, in peripheral blood leukocytes (PBLs) obtained from medical students at the time of examinations and at a baseline period approximately 1 month prior to the examinations. The level of messenger ribonucleic acid (mRNA) expression of both protooncogenes was significantly lower in PBLs obtained during examinations than in those from the baseline period. In addition, a significant decrease in the level of mRNA to the glucocorticoid receptor and gamma interferon was also found in the same preparations. The decrease in mRNA content of c-myc, c-myb, the glucocorticoid receptor, and gamma interferon in PBLs obtained from subjects during examinations is consistent with data from previous studies using the same model that have demonstrated a down-regulation of T-lymphocyte activation and proliferation in response to mitogens.

Host resistance to mycobacteria is compromised by activation of the hypothalamic-pituitary-adrenal axis.

Host resistance to the growth of Mycobacterium avium and Mycobacterium tuberculosis is controlled by a gene, termed Nramp1, that maps to chromosome 1 in mice. Activation of the HPA axis or treatment of macrophages from susceptible mice with corticosterone suppresses the expression of Nramp1 mRNA and results in an increased susceptibility to mycobacterial growth. In contrast, neither activation of the HPA axis nor treatment of macrophages from resistant mice with corticosterone results in an alteration in their resistance or suppression of Nramp1 expression. Investigation into the mechanism of the differential response of the macrophages to corticosterone indicated that differences were associated with the stability of the mRNA in macrophages from BCG-resistant mice. Thus, corticosterone induced the accelerated degradation of Nramp1 mRNA as well as mRNA of several other macrophage activation genes in macrophages from BCG-susceptible mice. Treatment of macrophages with corticosterone before the induction of Nramp1 resulted in the accelerated degradation of mRNA in macrophages from both resistant and susceptible mice. The Nramp1 gene product appears to protect the mRNA of macrophage activation genes from degradation induced by corticosterone by an iron-dependent mechanism.