ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins.

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.

Immune or normal gamma delta T cells that assist alpha beta T cells in elicitation of contact sensitivity preferentially use V gamma 5 and V delta 4 variable region gene segments.

In the current study, we confirmed previous findings suggesting that gamma delta T cells were involved in the successful adoptive cell transfer of contact sensitivity (CS) by alpha beta CS-effector T cells. In this study, we used hamster anti-mouse gamma delta-TCR mAb treatment of CS-effector T cells, followed by enrichment and removal of the gamma delta T cells with goat anti-hamster Ig-linked magnetic beads, or by addition of hemolytic rabbit C. This removal of gamma delta T cells abrogated adoptive cell transfers of CS, despite the presence of alpha beta T cells that are known to mediate CS. FACS analysis documented enrichment of gamma delta T cells rising from 1 to 2% of the starting cells, to 60 to 95% of the magnetic bead adherent cells. Adoptive cell transfer of CS was reconstituted by adding back to the alpha beta cells, highly enriched gamma delta cells attached to anti-gamma delta-TCR magnetic beads. Not only were gamma delta-enriched T cells from sensitized mice able to assist immune CS-effector alpha beta T cells, but gamma delta T cells from normal nonimmune mice also had CS-assisting activity, and furthermore, neither were MHC-restricted in this function. Thus, CS-assisting gamma delta T cells were present endogenously in normal mice without prior immunization, and acted without Ag specificity and without MHC restriction, to assist CS-effector alpha beta T cells. Similar studies, with hamster mAbs specific for V gamma and V delta portions of gamma delta-TCR, demonstrated that the gamma delta T cells that assisted the CS-effector alpha beta T cells preferentially expressed V gamma 5 and V delta 4 in their TCR. PCR analysis on extracted mRNA showed that V gamma 5 and V delta 4 gene segments indeed were rearranged and expressed in the sensitized and normal lymph nodes; and one-and two-color FACS analysis of magnetic bead-fractionated cells suggested that V gamma 5 and V delta 4 were expressed on the same T cells. In summary, these results demonstrated that V gamma 5+, V delta 4+, gamma delta T cells were needed to assist alpha beta effector T cells in the adoptive cell transfer of CS.

IL-3 dependence of a Thy-1lo, B220+, IL-3 receptor-positive antigen-specific DTH-initiating clone.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different antigen-specific Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This clone WP-3.27 produces an antigen-specific factor, OX-F, that acts in an Ag-specific manner to initiate DTH. The clone was phenotyped as a Thy-1+, B220+, CD3-, CD4-, CD8- cell. In this report, we further detail the characteristics of this unusual Ag-specific DTH-initiating cell clone. By flow cytometry analysis, WP-3.27 is Thy-1lo, Lyt-1+ (CD5+), but CD3-, TCR-alpha beta-, and TCR-gamma delta-. Moreover, WP-3.27 does not express surface immunoglobulins but expresses B220 (CD45RA), and also some macrophage markers such as Mac-1, F4-80, and MHC class II after gamma-IFN treatment. Interestingly, this clone also expresses IL-3 receptors (IL-3R) and not IL-2R. In addition to the Ag-specific DTH-initiating factor, WP-3.27 constitutively produces IL-3. Inhibition of proliferation of WP-3.27 with an anti-mouse IL-3 monoclonal antibody suggests that the clone WP-3.27 is IL-3-dependent, at least partially. WP-3.27 also constitutively produces IL-1 and IL-6, but not TNF-alpha. LPS activation of the clone resulted in a net increase of IL-1, IL-6, and TNF-alpha production. Thus, this Ag-specific DTH-initiating cell clone makes a unique set of cytokines. Northern blot analysis demonstrated that clone WP-3.27 transcribes mRNA encoding IL-1, IL-3, IL-6, and TNF-alpha, but not for TNF-beta (lymphotoxin). The nature of this unusual cell, which displays characteristics of more than one cell lineage, is discussed.

Induction of macrophage TNF alpha, IL-1, IL-6, and PGE2 production by DTH-initiating factors.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different, antigen-specific, Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This cell clone, WP-3.27, releases an antigen-specific factor (OX-F) that sensitizes mast cells such that specific antigen challenge will induce serotonin release which mediates the early phase of DTH. In normal mice contact sensitized with picryl chloride (PCl), a similar polyclonal factor (PCl-F) has a similar activity and is also known to bind to macrophages. Thus, we measured macrophage production of TNF alpha, IL-1, IL-6, and PGE2 in response to the hapten affinity-purified DTH-initiating factors OX-F and PCl-F. Both factors induced significant release of each cytokine and PGE2. The production of TNF alpha, IL-1, and IL-6 was measured by bioassays. Northern blot analysis showed rapid accumulation of cytokine mRNA (2-4 hr), while maximal production of PGE2 occurred at approximately 8 hr. These macrophage activating properties of OX-F and PCl-F were not due to contamination with LPS as determined by the low levels of LPS present in OX-F and PCl-F and by the failure of polymyxin B to inhibit factor-induced PGE2 and TNF alpha production. Also, macrophage activation was shown not to be due to the action of several lymphokines known to be produced by WP3.27. Separation of OX-F and PCl-F by preparative isoelectric focusing showed a similar pattern: there were two major peaks of PGE2-inducing activity observed for both factors (for PCl-F at pI of 2-3 and 5.0, and for OX-F at pI of 3.5-4 and 5.0), but not for a sham factor produced by WEHI-3 cells. The ability of DTH-initiating factors to rapidly induce macrophage cytokine release and PGE2 synthesis 4-6 hr later may suggest a role for these mediators during the respective early vascular and late cellular phases of inflammation in DTH.

An antigen-specific DTH-initiating cell clone. Functional, phenotypical, and partial molecular characterization.

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different Ag-specific Thy-1+ cells. An early-acting DTH-initiating cell in the lymphoid organs produces a circulating, Ag-specific factor that is functionally analogous to IgE antibody and initiates DTH by sensitizing the local tissue for release of the vasoactive amine serotonin. In picryl chloride (PC1) or oxazolone (OX) contact sensitivity, this DTH-initiating factor is called PC1-F and OX-F respectively, and is Ag-specific, but MHC-unrestricted. The phenotype of polyclonal DTH-initiating cells was recently shown to be unusual for an Ag-specific cell. The phenotype was: Thy-1+, Lyt-1+ (CD5), triple negative (CD4-, CD8-, and CD3-), B220+ (Ly-5, CD45RA), positive for IL-3 receptors, but not IL-2 receptors, and positive for antibodies that react with a putative constant or framework portion of DTH-initiating factors such as anti-PC1-F antibodies and 14-30 mAb. We report here the generation of an Ag-specific DTH-initiating cell clone from nude mice that were immunized and boosted by contact sensitization with OX. By flow microfluorometry analysis, this clone has a similar unique surface phenotype, and by in vivo assay has the same functional abilities, as polyclonal DTH-initiating cells. The clone produces Ag-specific OX-F that acts in an Ag-specific manner to initiate DTH. Moreover, specific cDNA probes and Northern blot analysis of the clone demonstrated that the Ag-specific DTH-initiating cells are Thy-1+, CD3-, and IL-3R+. Thus, DTH initiation is due to an Ag-specific lymphoid cell, that produces an Ag-specific factor, and that has a unique surface phenotype for Ag-specific cells; namely, Thy-1+, CD5+, sIg-, CD4-, CD8-, CD3-, CD45RA+, IL-2R-, and IL-3R+.

Chemical carcinogens induce cadmium resistance and activate metallothionein genes in cadmium sensitive S49 mouse cells.

Treatment of cadmium-sensitive (Cds) metallothionein-negative S49 mouse cells with two direct-acting chemical carcinogens (N-ethylnitrosourea or N-acetoxy-2-acetylamino-fluorene) or with u.v. radiation induced a large increase in phenotypically stable cadmium-resistant (Cdr) variants. In contrast, treatment with any of three agents which alkylate proteins (N-ethylmaleimide, iodoacetate, or phenylmethyl-sulfonyl fluoride) was without effect. Similarly, treatment with 2-acetylaminofluorene (a pre-carcinogen) or with 12-O-tetradecanoylphorbol-13-acetate (a tumor promoter) did not result in an increase in Cdr variants. Initial studies indicate that in many variants the metallothionein-I gene, the metallothionein-II gene, or both have been activated. Thus the induction of cadmium resistance in Cds cells is a potentially useful system to explore the activation of quiescent genes by carcinogens.

"sarc" sequence transcription in Moloney sarcoma virus-transformed nonproducer cell lines.

(3)H-labeled complementary DNA(sarc), complementary to the murine sarcoma virus (MSV)-specific portion of the Moloney MSV (M-MSV) genome, was prepared. M-MSV-specific RNA was then quantitated in the cytoplasm of several M-MSV-transformed, non-virus-producing, clonal NIH 3T3 cell lines. These lines, designated 71 N clones 5, 6, and 3, have been characterized previously by the degree to which they exhibit transformation properties and transcribe Moloney murine leukemia virus-related RNA (S. Salzberg and M. Green, J. Virol. 13:1001-1004, 1974; N. Tsuchida and M. Green, J. Virol. 14:587-591, 1974). By the criteria of cell morphology and agglutination by concanavalin A, cells of clone 5 are highly transformed, cells of clone 6 are almost normal in the sense that they resemble the parent NIH 3T3 cells, and cells of clone 3 are phenotypically intermediate. In the present study, the amounts of cytoplasmic MSV-specific RNA correlated well with the relative degrees of transformation of the cell lines, varying over 35-fold between the least transformed (clone 6) and most transformed (clone 5) lines. Superinfection of either clone 5 or clone 6 with Moloney murine leukemia virus resulted in a fivefold increase in the MSV-specific RNA in the cell cytoplasm. Evidence from (3)H-labeled complementary DNA:cell DNA hybridization studies indicated that the quantity of M-MSV-specific RNA in the nonproducer lines was not directly related to DNA provirus copy number in the cell DNA. Although clones 5 and 6 differ greatly in transformation characteristics and in MSV-specific RNA content, they each apparently contain about two copies of MSV-specific DNA sequence per haploid genome. Thus, factors such as site of provirus integration may be of primary importance in determining virus-specific transcription and cell transformation.