A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Structural identity of the TL antigen homologues derived from strain 2 and strain 13 guinea pigs

Strain 2 and strain 13 guinea pig thymocytes have been shown to bear a molecule that by several criteria appears to be a homologue of the murine TL antigen. The existence of a TL polymorphism in the mouse system as evidenced by TL- strains and various TL phenotypes in TL+ strains prompted a study to determine if a similar polymorphism could be demonstrated in the guinea pig system. By using two-dimensional gel electrophoresis, the thymocytes of a third inbred strain, DHCBA, were shown to bear a TL antigen, and the TL antigens of strains 2 and DHCBA were shown to give identical patterns of spots. A biochemical comparison of the strain 2 and strain 13 TL antigen heavy chains by tryptic and chymotryptic peptide mapping demonstrated that these molecules have identical peptides. Thus, no polymorphism could be demonstrated within the guinea pig TL system for the three inbred strains studied. Comparative tryptic peptide mapping of the guinea pig TL and class I B.1+S antigens demonstrated 43% homology, significantly higher than that reported for murine H-2 and TL antigens. These results provide suggestive evidence that the gene duplication giving rise to the genes determining the class I and TL antigens may have occurred more recently in the guinea pig than in the mouse.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

CTL and serologically defined antigens of B2m,H-3 region

The antigens of the B2m,H-3 region of 13 chromosome 2 congenic strains and seven inbred strains have been studied by using CML and serologic techniques. Nine patterns of cross-reactivity have been defined by CML assays. These results are in agreement with an extend previously described cross-reactivity studies. The reactivities of three monoclonal antibodies previously thought to be reacting with B2M-B are shown to differ: Ly-m11 and J-5 react with cells of strain B10-pa,at and clone 23 does not. Two H-3 region loci are hypothesized on the basis of CML and serologic activity: B2m and H-3. The CTL responses to the B2M antigens are H-2K restricted; the CTL responses to H-3 antigens are H-2D restricted. The restriction of the response to the H-3 antigen requires effector-target identity of the H-2D molecule but not the B2M molecule of the class I antigen. These loci have been separated by recombination from H-42 in the production of the congenic strain B10.FS-a. A gene order of B2m, H-3, H-42 is suggested.     

Immune responses of inbred guinea pigs against random terpolymers containing L glutamic acid and L alanine.

The immune responses of the inbred guinea pig strains 2 and 13 have been determined against random terpolymers of L glutamic acid and L alanine and a third amino acid. Strain 2 guinea pigs responded against GAT10, GAT20(LLD), GAT10(NO2)15, GAT4, and GAL10. However, strain 13 guinea pigs responded only against GAT10. The explanation offered is that strain 2 guinea pigs, which have the Ir-GA gene, recognize the polymers via random GA determinants present in sufficient concentration in all of the above polymers. However, strain 13 guinea pigs recognize the GAT10 via the Ir-GT gene, and reduction in the concentration of tyrosyl residues below 10 mole % by various procedures alters the concentration of available random GT determinants necessary for interaction with the gene product of the Ir-GT gene.     

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs.

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F₁ hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.     

γ-Glutamyl Cyclotransferase: A New Genetic Polymorphism in the Mouse (MUS MUSCULUS) Linked to LYT–2

Electrophoretically variant forms of gamma-glutamyl cyclotransferase have been identified in red cells of inbred mouse strains. Each inbred strain exhibited a major band of activity and a minor band that migrated more anodally. The polymorphism affects the migration of both the major and minor bands in a similar way. F1 hybrids between strains with fast forms (A/J) and strains with the slow forms (C57BL/6J) exhibited a four-banded pattern consistent with co-dominant inheritance. The patterns observed in backcross and F2 mice were consistent with the segregation of a pair of autosomal co-dominant alleles. Recombinant inbred strains and a congenic strain were used to show that the locus controlling gamma-glutamyl cyclotransferase (Ggc) is linked to Lyt-2, a lymphocyte alloantigen locus on chromosome 6, with an estimated map distance of 5.0 +/- 2.5 centimorgans.     

An erythrocyte and serum antigen with a specificity antithetical to the mouse C4 associated H-2.7 antigen

Repeated immunization of intra-H-2 recombinant strain A.TBR16 with lymphoid tissue from strain A.TBR13 produced an antiserum that agglutinated the erythrocytes from inbred strains of mice carrying the b, d, r, q, u, wr7, w13, w17, w19, and w21 haplotypes of the H-2 complex. The antiserum was negative with erythrocytes of strains carrying the haplotypes f, j, k, p, and s. This pattern of reactivity among fifteen H-2 haplotypes is unlike the strain distribution for any known H-2 erythrocyte antigen, and is exactly antithetical to the S region-controlled H-2.7 antigen. An examination of 12 intra-H-2 recombinant haplotypes mapped the genetic determinant controlling the new antigen to the IC or S regions of the H-2 complex. In addition, hemagglutination inhibition studies revealed the antigen was also expressed in serum and plasma. The serologic, genetic, and tissue distribution data suggest the gene controlling the newly defined antigen is allelic to the gene controlling the H-2.7 antigen.     

Serological studies on the major histocompatibility complex of new inbred strains of the guinea pig.

New inbred strains of guinea pigs, JY 1, JY 2, JY 3, JY 6, JY 9 and JY 10 have been established in this Institute. Serologic studies of guinea pig leukocyte antigens (GPLA antigens) were carried out in order to examine their major histocompatibility complex (GPLA complex). Antisera specific for Ia antigens were raised by cross-immunization of NIH strain 2 (NIH 2) and NIH strain 13 (NIH 13) guinea pigs, well known inbred guinea pigs. The sera identified four distinct Ia specificities, which were designated as Ia.2a, Ia2b, Ia.13a and Ia.13b. Six antigenic specificities different from the above Ia specificities were identified by sera obtained by appropriate immunization of the inbred guinea pigs and were designated as P.1, P.2, P.3, P.4, P.5 and P.6. Antigenic specificities of GPLA antigens recognized in inbred guinea pigs were : NIH 2 (Ia.2a, Ia.2b, P.1, P.2, P.O, P.4), NIH 13 (Ia.13a, Ia.13b, P.1, P.2, P.3, P.4), JY 1 (Ia.13a, Ia.13b, P.5), JY 2 (Ia.2b, Ia.13b, P.3, P.4, P.6), JY 3 (Ia.13a, Ia.13b, P.2, P.4, P.5), JY 6 (Ia.2b, Ia.13b, P.3, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6). The correspondence of these specificities to those already reported was discussed and the P.2 or P.4 was considered to be an additional specificity of GPLA antigens that have not been reported yet. As the new inbred strains of guinea pigs were thus found to possess characteristic GPLA complex, which differ from each other and from those of NIH 2 and NIH 13 strain, they should be useful for studies of roles of the major histocompatibility complex in the immune system.     

Characterization of macrophage adhesion molecule

Macrophage adhesion molecule (MAM), an abundant surface molecule which functions in the adhesion and spreading of guinea pig macrophages on surfaces, is characterized as a heterodimer of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). The density of MAM molecules is estimated at 630,000 per macrophage on the basis of quantitative binding of 125I-labeled monoclonal antibody. The glycopeptide subunits display microheterogeneity on isoelectrofocusing; the pI is 5.8-6.3 for gp160 (MAM-alpha) and 6.4-7.0 for gp93 (MAM-beta). A neutrophil gp160, gp93 molecule was shown to be indistinguishable from macrophage MAM on the basis of electrophoresis, isoelectrofocusing, and reactivity with 10 monoclonal antibodies. A related heterodimer of gp93 associated with a larger, antigenically different glycopeptide (gp180,gp93) was identified on circulating lymphocytes. Cumulative properties indicate that MAM is the guinea pig analogue of human Mo1 and mouse Mac-1.     

The major histocompatibility complex of the guinea pig. I. Serologic and genetic studies.

Serologic and genetic studies of the antigens which comprise the guinea pig MHC have demonstrated three distinct but linked genetic regions. Antisera to the B region were raised by cross-immunization of random-bred animals; this region controls antigens B.1, B.2, B.3, and B.4 which behave as alleles at a single locus and which resemble the products of the murine D or K region genes in their tissue distribution and molecular characteristics. Cross-immunization of inbred strain 2 and strain 13 animals, both of which bear the B.1 antigen, leads to sera which identify antigens which resemble the products of the I region of the murine MHC. Specific absorption experiments have demonstrated four distinct I region antigens. In addition to the B and I regions, inbred strain 2, strain 13, and some outbred animals bear an antigen (S.1) which is the product of a third genetic region and which also resembles the murine D or K region gene products in molecular size. The results of these studies should facilitate the use of the guinea pig as an experimental model for studies of genetic control of the immune response and the function of the histocompatibility-linked Ir genes.     

PCR扩增近交系大鼠微卫星位点DNA多态性的研究

本实验选取大鼠7条染色体上的微卫星位点合成了10对引物,利用聚合酶链反应（PCR）扩增技术对国内北京和哈尔滨等4家单位提供伯6个品系（SHR、SHRSP、LEW、RCS、WKY和F344）的8个近交系大鼠群体进行了DNA多态性分析的研究。结果表明：9个微卫星位点具有显多态性;不同品系个体之间具有多态性;同一群体不同个体之间除SHR（哈）的SMST位点和WKY（哈）的AGT位点出现一定的差异,其他均没有差异;不同地区同一品系的不同个体之间也存在一定的差异。该方法能有效地对近交系与杂交系、品系与品系、品系与亚系加以区分。因此,本实验为开展近交系大鼠遗传作图、基因定位和为实验动物的遗传背景监测提供可靠的信息,为大鼠遗传基因的研究提供了一个快速简例、特异准确的方法。     

微卫星DNA标记在近交系大鼠HFJ和MIJ遗传监测中的应用

目的 用24对引物对近交系HFJ和MIJ大鼠的微卫星位点进行多态性分析,并选用近交系Lewis和F344大鼠作为对照,进行比较分析.方法 用传统的酚-氯仿法分别提取4个近交系大鼠MIJ、HFJ、Lewis和F344 的基因组DNA,选取大鼠24个微卫星位点,通过PCR扩增,扩增产物经过非变性聚丙烯酰胺凝胶电泳和银染,根据电泳结果,比较分析4种品系近交系大鼠之间微卫星多态性.结果 4种品系及品系内不同个体的近交系大鼠在24个微卫星位点上的扩增产物均出现一个条带,MIJ和HFJ大鼠在品系间和品系内均表现为单态性,同Lewis 和F344的扩增结果比较,14个位点显示多态性,有10个位点显示单态性.结论 两个近交系大鼠品系MIJ和HFJ符合近交系要求,筛选出的14个多态性微卫星位点可用于有关近交系大鼠的遗传背景监测.     

Isolation of C4-binding protein from guinea pig plasma and demonstration of its function as a control protein of the classical complement pathway C3 convertase

A decay-accelerating factor of the classical complement pathway C3 convertase, C4b,2a, has been purified to homogeneity from guinea pig plasma by a 5-step procedure that includes 5% polyethyleneglycol-4000 (PEG-4000) precipitation, Sepharose 6B gel filtration, heparin-Sepharose chromatography, DE-52 anion exchange chromatography, and Sepharose-C4gp affinity chromatography. The protein elicited a monospecific antiserum in a rabbit and was found with the Mancini technique in both normal and C4-deficient guinea pig plasma at a concentration of 60 microgram/ml. The purified protein gave a single stained band of 550,000 m.w. on SDS-PAGE under nonreducing conditions and a single band of 72,000 m.w. with reduction and alkylation. On the basis of its m.w. and subunit structure, ability to bind to a C4 affinity column, and ability to regulate the classical C system by accelerating the decay of the classical C3 convertase this protein represents the guinea pig analog of the human C4-binding protein.     

Restriction by the major histocompatibility complex of antigen-induced T lymphocyte proliferation in new inbred guinea pig strains.

Employing new inbred guinea pig strains, JY 1, JY 2 and JY 3, established in this Institute in addition to strains 2 and 13, the authors investigated histocompatibility restriction in macrophage-T lymphocyte interaction. These five strains are known to possess distinct major histocompatibility complex (MHC) gene profiles (1, 2). This fact was supported by our results concerning the mixed leukocyte reaction (MLR) and cytotoxicity test with alloantisera. Using various combinations of T lymphocytes and peritoneal exudated cells (PECs) from these strains, in vitro proliferative responses of T lymphocytes from BCC-immune animals to PPD-pulsed normal PEC were tested. Successful activation of T cell response was observed not only in syngeneic combinations but also in allogeneic combinations among strains JY 1, JY 3 and strain 13 which share common Ia antigens detected by strain 2 anti-strain 13 alloantiserum. Because JY 1 and JY 3 seem to share a common B antigen differing from strain 13, it was suggested that identification in the I region of MHC is sufficient for effective antigen-presentation by the macrophage. Although a part of Ia is shared, no T lymphocyte activation was observed in the combination between JY 2 and JY 1 or JY 3, whereas strong MLR occurred in these allogeneic combinations. At the present stage of the study, it can be said that disparity in the part(s) of Ia antigens which is responsible for strong MLR cannot lead to effective T cell-macrophage interaction. These results support the concept that functional activation of primed, proliferating T lymphocyte requires the participation of gene products of macrophages coded for by the I region in MHC. By employing JY 1, JY 2 and strain 2, which appear to possess distinct B and Ia antigens, it was shown that the T lymphocyte and macrophage interactions essential for mitogen-induced T lymphocyte proliferation are not restricted by histocompatibility.     

Mechanisms of C-type viral leukemogenesis. I. Correlation of in vitro lymphocyte blastogenesis to viremia and leukemia.

Various inbred strains of mice respond immunologically to genetically transmitted ecotropic C-type viruses. Part of this response is T cell blastogenesis with type specificity for the viral envelope glycoprotein gp71. Of those nonviremic, nonleukemic strains, and F1 crosses examined, in which virus expression occurs early in life, gp71-specific blastogenic T cells were detected within the first 2 months of age and temporally preceded the development of a humoral immune response. However, in the viremic, highly leukemic strain of AKR mice, gp71-specific T cell blastogenesis in vitro was readily detectable throughout the preleukemic phase, the first 5 months of age. In appropriate F1 crosses and backcrosses, the persistent in vitro blastogenic response segregated with viremia and leukemia. These data suggest that in vivo T cell stimulation by endogenous viral gp71, caused by viremia, may contribute to virus-induced leukemogenesis in mice.     

Mitogenic factor from inbred guinea pigs. I. Isolation of the factor

Methods are described for the reproducible elicitation of mitogenic factor (MF) from antigen-sensitive lymphocytes of inbred strains of guinea pigs. The use of inbred animals minimizes complications due to histocompatibility factors. Each of several antigens tested was effective. Mitogenic factor is released in vitro as early as 6 hr after stimulation of lymphocytes by antigen. It was obtainable from serum-free cultures in which medium RPMI-1640 was used; this should facilitate isolation of MF. The addition of 5 mMl-cysteine to cultures substantially improved the yield of MF. MF was obtained from cultures of lymph node cells of highly purified small lymphocytes, which indicates that the small lymphocyte is the source of MF in the guinea pig. It was shown that MF can induce mitosis as well as blast transformation in non-immune lymph node cells. MF from a given strain of guinea pig is capable of stimulating lymphocytes of another strain.     

Guinea pig lymphocyte antigen molecules Ia.3,5 and Ia.8 share structural homology expected for alleles

Because of the lack of I region recombinant guinea pig strains, rigorous comparative chemical analyses of guinea pig Ia alloantigens have been utilized to gather structural information that would allow tentative assignment of I region alleles. In this study, the B/Lac Ia.8 molecule was chemically compared to the strain 13 Ia.3,5 molecule and the strain 2 Ia.2 and Ia.4,5 molecules. SDS-PAGE, IEF, and Cleveland peptide mapping demonstrated a significant degree of homology between the Ia.8 and Ia.3,5 alpha-chains. HPLC mapping of trypsin/chymotrypsin cleavage products revealed an overall coincidence of peptides of 57 and 68% for the comparisons of the Ia.8 and Ia.3,5 alpha- and beta-chains respectively. In contrast, comparisons of Ia.8 and Ia.2 alpha- and beta-chains exhibited a significantly lower degree of similarity--39 and 47% respectively. The degree of homology seen in the Ia.8 and Ia.3,5 comparisons is consistent with those values seen in comparisons between the Ia.3,5 and Ia.4,5 molecules (putative allelic products of the guinea pig I region) and with those values obtained for allelic products of the mouse H-2 system. The results of this investigation strongly suggest that the Ia.8, Ia.3,5, and Ia.4,5 molecules are the products of alleles at the Ia alpha- and beta-chain loci.     

Malocclusion of the premolar and molar teeth in the guinea pig.

Malocclusions of the premolar and molar teeth of inbred Strain 2/N and Strain 13/N and outbred Dunkin-Harley guinea pigs were examined. A higher incidence of malocclusion observed in the inbred strains suggested a genetic basis for the disorder.     

Biochemical characterization of the molecules reactive with Qa-6 antiserum and the monoclonal antibody 20-8-4: evidence for structural similarity with the Qa-2 molecule

The Qa-6 alloantigen and the molecule that crossreacts with the monoclonal antibody (mAb) 20-8-4 have been shown to be serologically distinct from the Qa-2 alloantigen by strain distribution and tissue distribution, respectively. In this report, we address the biochemical relationships among Qa-2, Qa-6, and the 20-8-4 cross-reactive molecule by using immunoprecipitation and polyacrylamide gel electrophoresis. Each of these molecules had an apparent m.w. of approximately 41K and was associated on the cell surface with beta 2-microglobulin. Removal of N-linked oligosaccharides with endoglycosidase F reduced their apparent m.w. to approximately 33K to 34K. The determinants recognized by anti-Qa-6 and mAb 20-8-4 were shown to reside on the same molecule(s) precipitated by anti-Qa-2 sera by immunodepletion experiments. The mAb 20-8-4 was also shown to preclear the molecules detected by the Qa-6 and Qa-2 antisera. Two-dimensional gel electrophoresis analysis demonstrated complete co-migration of the approximately 41K molecules detected by the three antibodies. By peptide map analysis with V8 protease, all three molecules appeared identical. Also, the determinant recognized by Qa-6 antiserum co-modulated with that recognized by the anti-Qa-2 mAb D3.262. Taken together, these results demonstrate that the molecules recognized by these three antisera and/or mAb are biochemically indistinguishable. These data, in conjunction with the serologic and genetic findings suggest that mAb 20-8-4 recognizes a molecule that is biochemically similar and possibly identical to the Qa-2 antigen. Moreover, although the genetic, serologic, and biochemical data demonstrate that Qa-6 is not controlled by the Qa-2 locus, but rather by a gene telomeric to Qa-2, the molecule bearing the Qa-6 determinant is very similar, if not identical, to the Qa-2 molecule. Several possible explanations for these discrepancies are discussed.