Patricia P. Jones

Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and Ia Antigens

Advances in somatic cell hybridization techniques have made it possible to generate hybrid cell lines producing monospecific antibodies directed at desired antigenic determinants (1). In this paper a modification of the cell fusion procedure (2,3) was used to recover stable hybrid cell lines secreting IgG antibodies to: (a) mouse major histocompatibility complex (MHC) alloantigens (H-2K and I-A); and (b) mouse immunoglobulin (Ig) allotypes (Ig-1b, Ig-5a, and Ig-5b).

Nomenclature for the major histocompatibility complexes of different species: a proposal.

The major histocompatibility complex (MHC) has been given different names in different species (Klein 1986). It is designatedH-2 in the mouse, HLA in humans, B in the domestic fowl, RT1 in the rat, and Smh in the mole rat. In most other species that have been studied, the MHC is referred to by the LA symbol (for lymphocyte or leukocyte antigen), prefixed by an abbreviation of the species’ common name. Thus, it is called ChLa in the chimpanzee, GoLA in the gorilla, RhLA in the rhesus macaque, RLA in the rabbit, BoLA in the domestic cattle, SLA in the pig, and so on. This practice has two problems associated with it. First, MHC products are expressed on many other tissues in addition to lymphocyte or leukocyte (and lymphocytes express many other antigens in addition to those controlled by the MHC) and their antigenicity is secondary to their biological function. Second, the use of common names to identify a species is a potential source of confusion. Common names are notoriously vague and imprecise. The designation “lemur”, for example, can refer to any of the genera Lemur, Hapalemur, Varecia, Lepilemur; Avahi, Propithecus, and Indri, of which only the first four belong to the family Lemuridae; the last three are members of the family Indriidae. A “bushbaby” can be a Galago, Otolemur, or Euoticus. A “mouse” could be a Notomys, ylcomys, Uranomys, Pogomys, Chiruromys, Chiropodomys, Neohydromys, and so on. Obviously, common names not only fail to identify the species appropriately, they often do not even identify the genes or the family. If the trend in choosing common names for MHC symbols were to continue, chaos would soon ensue because we can expect MHCs in many different species to be identified in the future.

Variable synthesis and expression of E β and A e (E β ) Ia polypeptide chains in mice of differentH-2 haplotypes

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice of thed, k, p, andr haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, Eα and Ae (Eβ). Cells from mice of theb ands haplotypes fail to synthesize Eα chains but do synthesize Ae chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of thef andq haplotypes fail to synthesize either the Ae or Eα polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact Eα:Ae complex are consistent with the theory that both the Ae and Eα polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics ofI-region-controlled functions are discussed.

Detection of a common polypeptide chain in I--A and I--E sub-region immunoprecipitates.

Two-dimensional polyacrylamide gel electrophoresis of I-A and I-E sub-region antigens has indicated that all Ia antigen immunoprecipitates contain three distinct polypeptide chains. Two of these molecules show haplotype-specific differences in electrophoretic mobility, while the mobility of the third chain (mol. wt 31,000 daltons) is the same as for I-A or I-E sub-region precipitates from all six H-2 haplotypes tested. The mobility of this invariant la-associated chain (provisionally called Ii) is also not affected by different genetic backgrounds. Ii is found uniquely in Ia antigen immunoprecipitates; its lack of detectable polymorphism suggest that it is precipitated because it binds specifically to the polymorphic I-region products. Ii does not label with 125I using lactoperoxidase, and it is not yet known whether Ii is part of the native Ia molecule on the cell surface, or whether it binds to Ia antigens during extraction.

A rapid sensitive assay for specific protein synthesis in cells and in cell-free translations: Use of Staphylococcus aureus as an adsorbent for immune complexes

Abstract Formalin-fixed, heat-inactivated Staphylococcus aureus Cowen Type I (SAC) (Kessler, S. W. (1975) J. Immunol. 115 , 1617–1624) has been used to isolate antibody-bound tyrosine aminotransferase (EC 2.6.1.5) from radioactively labeled cell-free extracts and translation reactions of polysomes and poly(A)-containing messenger RNA. Under our conditions, SAC immunoprecipitation of cell-free extracts generated a radioactive background in control serum precipitations of 0.1%, an average of fivefold lower than backgrounds obtained by direct and indirect antibody precipitations. It is shown that the major source of the background, which limits sensitivity of the assay, comes from the amounts of antiserum and bacterial adsorbent in the assay. Procedures are presented to provide low backgrounds in more general applications of the precipitation method. From these experiments, SAC immunoprecipitation is shown to be a rapid, economical method to assay for specific protein synthesis under a variety of experimental conditions with noise levels much lower than with other standard methods.

The invariant chain of murine ia antigens: Its glycosylation, abundance and subcellular localization☆

Abstract The properties of the invariant Ia antigen-associated polypeptide chain (I i ) have been examined. Two-dimensional gel analysis of Ia antigens immunoprecipitated from tunicamycin-treated murine spleen cells has been employed to identify the non-glycosylated polypeptide precursors for I i as well as for the I-A and I-E Subregion controlled chains. The results indicate that I i contains two N-linked carbohydrate units. Evidence is also presented that I i does not bind adventitiously to Ia during extraction procedures and that a pool of free I i may exist in the cell which is not bound to the polymorphic chains. Immunoprecipitation of Ia antigens from only the cell surface-expressed subset of Ia molecules or from isolated plasma membrane shows I i to be absent from these preparations. Since I i is found in cellular membranes but not free in the cytosol, it seems likely that I i is associated with the polymorphic Ia chains in the intracellular membranes but not on the cell surface.

Calcineurin Negatively Regulates TLR-Mediated Activation Pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-κB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-κB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IκB-α degradation, MAPK activation, and TNF-α production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.

Calcineurin inactivation leads to decreased responsiveness to LPS in macrophages and dendritic cells and protects against LPS-induced toxicity in vivo

Microbial components such as lipopolysaccharide (LPS) bind to Toll-like receptors (TLRs) and activate innate and inflammatory responses. Responses to LPS and other microbial components are limited by the activation of negative feedback mechanisms that reduce responsiveness to subsequent LPS exposure, often termed LPS tolerance. Our laboratory has previously shown that calcineurin, a phosphatase known for its activation of T cells via NFAT, negatively regulates the TLR pathway in macrophages; consequently, calcineurin inhibitors (FK506 and cyclosporin A) mimic TLR ligands in activating the TLR pathway, NF-KB, and associated innate and inflammatory responses. This study investigated the physiological consequences of calcineurin inactivation for LPS-induced inflammatory responses in vitro and in vivo using two models: calcineurin inhibition by FK506 (tacrolimus) and myeloid cell-specific calcineurin deletion. Activation of dendritic cells and macrophages with FK506 in vitro was shown to induce a state of reduced responsiveness to LPS (i.e. a form of LPS tolerance). Similarly, macrophages from FK506-treated mice or from mice in which the calcineurin B1 (CnB1) subunit was conditionally knocked out in myeloid cells were found to have diminished LPS-induced inflammatory responses. In addition, mice with CnB1-deficient myeloid cells and mice undergoing FK506 treatment showed improved survival and recovery when challenged with high doses of systemic LPS compared to controls. These results demonstrate that inactivation of calcineurin in macrophages and other myeloid cells by inhibition or deletion can induce a form of LPS tolerance and protect the host from LPS toxicity in vivo.

Revised nomenclature of mouse H-2 genes

The H-2 nomenclature was last revised in 1974 (Klein et al. 1974; Shreffler et al. 1974). Since then it has gradually gone out of step with the advances in molecular biology of the H-2 complex as well as the rules for genetic nomenclature established by the International Committee for Mouse Genetic Nomenclature (Lyon 1989). Furthermore, multiple symbols are currently in use designating the same H-2 genes or their products. The present proposal rectifies the existing inconsistencies in the H-2 nomenclature while preserving as much as possible from the previous proposals.

Changes in protooncogene expression correlated with general and sex-specific differentiation in murine primordial germ cells

Primordial germ cells (PGCs) of the mouse undergo key developmental transitions during embryonic days 12-15. On day 12 they complete migration into the gonads. They cease mitotic proliferation on day 13 and subsequently enter sex-specific pathways of development. The molecular mechanisms controlling these transitions are poorly understood, yet they are crucial to production of normal gametes later in life. We have used the polymerase chain reaction (PCR) to directly compare levels of expression of several protooncogenes proposed to be involved in control of cell proliferation and differentiation in proliferating and differentiating PCGs of both sexes over a 4 day time course. We report here that mRNA levels for nuclear protooncogenes c-myc, c-fos, and c-jun increase dramatically in both sexes from little or no detectable expression on day 12 to high expression on days 13-15. We observe c-kit message on day 12 in combined PGCs of both sexes, in female but not male PGCs on day 13, and in both sexes on day 14, c-kit mRNA is undetectable on day 15 in either sex, c-mos is not expressed at detectable levels on day 12 in either sex, but increases gradually in female PGCs to very high levels on day 15. In male PGCs, c-mos is expressed at high levels on days 13-15. Our results are consistent with a role for protooncogenes c-myc, c-fos and c-jun in mediating the initial differentiation of PGCs of both sexes that occurs upon colonization of the gonad. Because c-kit and c-mos are expressed differentially in male and female day 13-15 germ cells, they may play roles in initiating or mediating progress along the sex-specific pathways of development that PGCs embark upon at this time.