Downregulation of HLA class I antigens in HIV-1-infected cells.

By means of indirect immunofluorescence analysis we investigated the effect of HIV-1 infection on HLA class I surface antigens. We report here that in CD4+ HeLa cells, in H9 cells, and in peripheral T lymphocytes HLA class I antigens are downregulated following infection with HIV-1. The downregulation is effected at a posttranscriptional level since the amounts of HLA class I specific mRNA are similar in infected and uninfected cells. This phenomenon is not only correlated with the state of infection, that is, the presence of P24 of HIV-1 in the cells, but also depends on the time of infection. Upon HLA class I downregulation by HIV infection, the specific lysis of peripheral blood cells by allogeneic CTL is reduced.

Tumor necrosis factor amplifies measles virus-mediated Ia induction on astrocytes.

We describe the induction of Ia on cultured astrocytes by measles virus and the amplification of this induction by tumor necrosis factor (TNF). Measles virus induces Ia on rat astrocytes by direct interaction with these cells. TNF does not induce significant levels of Ia at any dose from 1 to 10,000 units/ml. As little as 10 units of TNF per ml, however, amplifies Ia-inducing signals generated by measles virus in astrocytes. In contrast, TNF and measles virus induce class I major histocompatibility complex (MHC) antigens, when applied individually, and TNF amplification of measles virus class I MHC induction is not apparent. The induction of either Ia or class I MHC antigens on rat astrocytes by measles virus does not depend on glial-derived soluble factors generated during infection. Since brain cells are normally lacking MHC antigens upon which T cells depend for interaction with antigen presenting cells, these data indicate that the ability of measles virus to directly stimulate MHC antigen expression and the ability of TNF to amplify Ia expression locally in the brain may be important in initiating cell-mediated immune response to viral infection.

Cellular oncogenes and lymphocyte activation.

In normal mouse splenic B and T cells at least two cellular proto-oncogenes are expressed, i.e. c-myc and c-fos. The expression of c-myc depends on the activation of the cells, but not on subsequent growth. C-fos gene expression appears to be induced by the manipulation involved in preparation of single cell suspensions from spleens. In that respect, c-fos gene expression does not qualify as being significantly involved in transition from G O to S phase while expression of c-myc seems to be correlated with some early events of cell activation leading to growth competence. The kinetics and extent of c-myc gene expression vary with the mitogen used and the type of lymphocyte investigated as examplified by T cells and subpopulations thereof. The expression of both proto-oncogenes in normal mouse spleen cells is finely regulated by an interplay of transcriptional and post-transcriptional control mechanisms. These mechanisms operate differently for the two genes and independently from one another. They also change in predominance at various times, again independently from one another. While we have no evidence that c-fos has significance for the activation of lymphocytes, c-myc is a good candidate for being involved. Thus studies by Susan Corey and collaborators on transgenic mice which constitutively express c-myc in cells of the lymphocyte lineage, indicate that this lineage is profoundly affected. Among others, the effects concern the balance between proliferation and maturation and a constitutive high level of Ia expression, normally only observed in activated cells. Constitutive high expression of c-myc in B cells of these transgenic mice also makes them prone to leukemia possibly due to a series of subsequent events. These last findings also provide an explanation for the need for a very finely tuned regulation of c-myc gene expression as it is here described.

Kinetics of cellular oncogene expression in mouse lymphocytes. II. Regulation of c-fos and c-myc gene expression.

Newly isolated lymphocytes from mouse spleens express the c-fos oncogene even in the absence of mitogen with maximal mRNA levels 60 min post preparation of single cell suspension, whereas c-myc mRNA levels increase only after mitogenic stimulation with maximal mRNA levels 6 h post stimulation. The half-lives of c-fos mRNA are generally very short; they increase from 14 min (after 30 min of culture) to 70 min (after 2 h of culture). The half-lives of c-myc mRNA decrease from 50 min (at 2 and 6 h post stimulation with concanavalin A) to 12 min (at 48 h post stimulation). The c-fos gene transcription is already turned on in time-0 lymphocytes 10 min after disruption of the organ structure of the spleens and is down-regulated after 2 h and later. In nuclear run-on experiments with nonstimulated lymphocytes there is already significant transcription of the first exon of c-myc, but almost no elongation of the transcript to exon 2 and 3. In concanavalin A-treated lymphocytes elongation is stimulated about 5-fold within 6 h and returns to background levels at 48 h post stimulation. The nuclear run-on analyses of nonactivated lymphocytes showed a signal for RNA complementary to c-myc mRNA detected with a probe specific for the exon 1/intron 1 boundary of c-myc, which disappeared with increasing time of concanavalin A stimulation. This anti-sense transcription may play a role in regulating the elongation of c-myc transcripts.

Regulation of IL 2 expression in mitogen-activated murine T lymphocytes.

In mitogen-stimulated mouse spleen cells, the IL 2 gene is only transiently expressed with maximal mRNA steady state levels between 6-14 h post stimulation with Concanavalin A (Con A). This is also reflected by the kinetics of IL 2 release into culture supernatants. Con A-stimulated L3T4+ and Lyt2+ T cell subpopulations express the IL 2 gene and produce IL 2 similarly. The half-life of IL 2 mRNA is only 30 min, but can be prolonged significantly by cycloheximide. At later post stimulation times IL 2 gene transcription is reduced, as indicated by the reduced effect of cycloheximide. IL 2 gene expression is not influenced by added IL 2 or IFN-gamma.

Transcriptional control of mu- and kappa-gene expression in resting and bacterial lipopolysaccharide-activated normal B cells.

When murine resting B cells are polyclonally stimulated by bacterial lipopolysaccharide (LPS) in vitro for a short period of 4 days, they are activated to DNA synthesis and cell division, and they also differentiate to immunoglobulin (Ig)-secreting plasma cells. These two events are accompanied with several qualitative changes at the Ig mRNA level: the disappearance of delta mRNA after stimulation, the switch from membrane to secretory form of mu-mRNA, and the late appearance of IgM joining chain (J-chain) mRNA. There is also a quantitative increase of Ig-gene expression at the level of: Ig gene transcription, mu-, kappa- and J-chain mRNA accumulation, and Ig translation and secretion. A comparison of Ig transcription rates before and in the course of LPS stimulation, as determined by in vitro transcriptional run-on assays, has shown that there is a large increase of the RNA polymerase density on both mu- and kappa-loci (30-60-fold), which is quantitatively comparable with the accumulation of both mu- and kappa-mRNAs at the steady state mRNA level. These data therefore suggest that former results obtained with tumor cells regarding post-transcriptional control of Ig gene expression do not reflect the physiological behavior of normal B cells with respect to the molecular events of B cell triggering. We also propose that additional molecular events such as RNA processing and the transcriptional activation of J-chain gene might be essential for controlling the maximal transcriptional rate across the Ig loci.

Kinetics of cellular oncogene expression in mouse lymphocytes. I. Expression of c-myc and c-ras-Ha in T lymphocytes induced by various mitogens.

Murine splenic T lymphocytes display maximal cellular myc gene (c-myc) expression already 3 h after concanavalin A stimulation and subsequent down-regulation before the onset of DNA synthesis. Stimulation by leucoagglutinin in the presence or absence of interleukin 2 leads to only low initial levels of c-myc-specific RNA which, however, increase later on. A similar pattern of c-myc expression is shown by the Lyt-2+ T cell subpopulation stimulated with either concanavalin A or leucoagglutinin in the presence of interleukin 2. Although [3H]thymidine incorporation was identical, the leucoagglutinin-stimulated Lyt-2+ T cells were void of any demonstrable c-myc-specific RNA at 3 h post-stimulation. Thus, the kinetics of c-myc expression in mouse T lymphocytes are not at all uniform, but depend on the mitogen and the subpopulation. In contrast, levels of c-ras-Ha-specific RNA were always low at early times, always increased towards the onset of DNA synthesis and down-regulation was not observed.

Behaviour-associated alopecia areata in mice.

In 5 different mouse strains, we observed alopecic lesions most commonly in male breeding animals kept with C57BL females. Alopecic areas were located most frequently on the head, but were also found over the shoulders, the back and the pelvic regions. Observations gained through time-lapse photography indicate the cause is a slight increase in self-grooming and a dramatic increase in allogrooming by their female partners. The increased frequency of lesions of males kept with C57BL females suggests that the increased grooming activity may be genetically determined.

High yields of DNA-transfer into mouse L-cells by electropermeabilization.

DNA transfection in mouse L-cells was performed by means of the electropermeabilization technique (U. Zimmermann, G. Pilwat, and F. Riemann, Z. Naturforsch. 29, 304 (1974)). The plasmid pSV 2-neo used leads to neomycin-resistance in stably transfected L-cells. Optimized conditions resulted in high yields of clones at relatively low DNA concentration. The influence of temperature during pulse application and during the subsequent resealing process as well as the field parameters and medium composition are discussed.

IgM RNA switch from membrane to secretory form is prevented by adding antireceptor antibody to bacterial lipopolysaccharide-stimulated murine primary B-cell cultures.

Bacterial lipopolysaccharide (LPS) induces proliferation of resting primary murine B lymphocytes and their differentiation into Ig-secreting cells. This is accompanied by an increase in the rate of Ig gene transcription and the accumulation of mu heavy chain secretory mRNA. Specific antiantigen receptor antibody (anti-mu) induces resting B cells to proliferation but not differentiation. Upon addition of both LPS and anti-mu to cultures, resting B cells again proliferate but do not differentiate. RNA transfer blots of the Ig mRNA 2 days after induction with LPS/anti-mu show a specific deficiency of the 2.4-kilobase (kb) mu secretory mRNA, whereas the levels of the 2.7-kb mu membrane and 1.2-kb kappa light chain mRNAs are as high as in cells treated with LPS alone. Between days 3 and 4 after treatment with both reagents, reductions of mu membrane and, to a smaller extent, kappa mRNA become apparent. As measured by nuclear run-on transcription experiments at day 2, the transcription rates of Ig mu and the Ig kappa transcription units are equal in both induction experiments. Only at later stages do the LPS/anti-mu-treated cells transcribe Ig genes at a lower rate. Thus, the anti-mu treatment, drastically reducing the mu secretory mRNA production at early stages, represents a negative regulation occurring primarily at the posttranscriptional level.

Expression of endogenous retroviral glycoprotein 70 by antigen-activated cytotoxic and suppressor T lymphocytes of nice.

Concanavalin A-stimulated murine spleen cells and antigen-stimulated B lymphocytes of normal mice express an antigen that reacts with goat antiserum against glycoprotein (gp) 70. Structural analysis of this antigen characterizes it as endogenous viral gp70 that is most likely of xenotropic origin. Activated nonspecific T suppressor cells and cytotoxic T lymphocytes express endogenous viral gp70, whereas nonactivated mouse T or B lymphocytes do not. The presence of endogenous retroviral gp70 is thus a novel marker for activated mouse lymphocytes in general.

In vitro differentiation of F-9 teratocarcinoma cells does not induce expression of endogenous retroviral glycoprotein.

Undifferentiated F-9 teratocarcinoma cells derived from 129/J mice are induced in vitro to express several differentiation markers. Neither undifferentiated nor differentiated F-9 cells express endogenous retroviral glycoprotein (gp70), although the latter can be productively infected with exogenous retroviruses. This is discussed in context with previous findings that all antigen-activated lymphocytes of all mice express endogenous retroviral gp70.

A modified absorption-reduction method to detect virus-specific hemagglutination inhibiting and neutralizing IgM antibodies.

IgG antibodies are preferentially absorbed by protein A-coupled sepharose beads while most of the IgM and IgA antibodies remain in solution. Even relatively low amounts of virus-specific IgM antibodies can then be detected unequivocally by a decrease of the antibody titer following the treatment with reducing agents. Ethandithiol proved superior to 2-mercaptoethanol in combination with neutralization assays.

Chemical characterization of macrophage cytotoxicity factor, macrophage migration inhibitory factor, T-helper cell-replacing factor and colony-stimulating factor from culture supernatants of concanavalin A-stimulated murine spleen cells.

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatograhy of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.