Horizontal transfer of oncogenes by uptake of apoptotic bodies.

Tumor formation involves the accumulation of a series of genetic alterations that are required for malignant growth. In most malignancies, genetic changes can be observed at the chromosomal level as losses or gains of whole or large portions of chromosomes. Here we provide evidence that tumor DNA may be horizontally transferred by the uptake of apoptotic bodies. Phagocytosis of apoptotic bodies derived from H-ras(V12)- and human c-myc-transfected rat fibroblasts resulted in loss of contact inhibition in vitro and a tumorigenic phenotype in vivo. Fluorescence in situ hybridization analysis revealed the presence of rat chromosomes or of rat and mouse fusion chromosomes in the nuclei of the recipient murine cells. The transferred DNA was propagated, provided that the transferred DNA conferred a selective advantage to the cell and that the phagocytotic host cell was p53-negative. These results suggest that lateral transfer of DNA between eukaryotic cells may result in aneuploidy and the accumulation of genetic changes that are necessary for tumor formation.

Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells.

Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76+/-13%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19+/-19% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26+/-14%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.

Up-regulation of CCR5 expression in the placenta is associated with human immunodeficiency virus-1 vertical transmission.

The role of placenta in vertical transmission is not yet fully understood. A protective role of the placenta during gestation is suggested by the finding that caesarian sections reduce the risk of transmission of human immunodeficiency virus (HIV)-1 from mother to child three- to fourfold. Here we investigated whether the immunological milieu of the placenta might be important in HIV-1 transmission. In situ imaging of immunohistochemically stained placenta sections and reverse transcriptase-polymerase chain reaction demonstrated a fourfold increase in CCR5:CXCR4 expression ratio in placentae from transmitting women compared to placentae from nontransmitting women. This chemokine receptor repertoire was consistent with an up-regulation of interleukin-4 and interleukin-10 expression in placentae from nontransmitting placentae compared to transmitting placentae. In situ imaging demonstrated that CCR5 and CXCR4 were expressed on placental macrophages and lymphocytes but not in trophoblasts. Simultaneous immunofluorescence/ultrasensitive in situ hybridization for HIV-1 gag-pol mRNA revealed that HIV-1 infects primarily CXCR4-expressing cells in placentae from nontransmitting women whereas predominantly CCR5-expressing cells were infected in placentae from transmitting women. These data are consistent with transmission of a homogeneous population of nonsyncytium-inducing HIV-1 isolates that use CCR5 as co-receptor.

Normalization of immune activation in lymphoid tissue following highly active antiretroviral therapy.

Although significant progress has been made in understanding immune reconstitution in peripheral blood following highly active antiretroviral therapy (HAART), less is known about immune changes in lymphoid tissue. Here, the expression of cytokine proteins (interferon gamma [IFN-gamma], interleukin [IL]-2, IL-4, IL-10, IL-1alpha, and IL-1beta) and surface antigens (CD4, CD8, CD1a, CD68) as well as cellular proviral HIV-1 DNA were determined in sequential tonsil biopsies before and at 4, 12, and 48 to 56 weeks posttherapy by quantitative in situ image analysis and fluorescent in situ 5;-nuclease assay (FISNA). Despite plasma virus suppression, a fraction of tonsil cells harbored pro-viral DNA for up to 1 year. A fourfold to eightfold increase in CD8+ T cells in tissue compared with seronegative controls and an increased frequency of CD1a+ dendritic cells prior to HAART reached control levels at week 56. The frequency of IFN-gamma expressing cells was 10-to 15-fold higher than controls before therapy and was comparable with findings in seronegative controls by week 56. Elevated baseline expression of IL-1alpha and IL-1beta was reduced by week 4 but IL-1alpha levels remained elevated in 1 of 3 patients at week 56. These findings suggest that with effective viral suppression the immune system in tissue may return to a more resting state.

Human immunodeficiency virus type 1 infection is associated with significant mucosal inflammation characterized by increased expression of CCR5, CXCR4, and beta-chemokines.

Mucosal inflammation is characterized by increased expression of proinflammatory cytokines and chemoattractant chemokines, resulting in infiltration of immunocompetent cells. This study compared the degree of mucosal inflammation in human immunodeficiency virus type 1 (HIV-1)-infected gut mucosa with that in tissue samples from subjects with inflammatory bowel disease (IBD) and from healthy seronegative control subjects. Gut mucosal biopsy specimens were immunohistochemically stained and were evaluated by in situ imaging. There was significantly increased expression of HIV-1 coreceptors CCR5 and CXCR4, beta-chemokine RANTES, and macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, as well as increased numbers of T cells in lamina propria of HIV-1-infected patients. The results were similar in patients with IBD and in HIV-1-infected patients, suggesting increased inflammation in the colon of HIV-1-infected patients. To further investigate the effect of inflammation in HIV-1-infected lamina propria, treatments that reduce immune activation in lamina propria must be evaluated.

Functional gene transfer of HIV DNA by an HIV receptor-independent mechanism.

HIV-1 enters target cells mainly via binding to CD4 and its coreceptors. The presence of HIV-1 in CD4- cells suggests, however, that there exist other mechanisms for viral entry. Here it is reported that HIV-1 DNA may be transferred from one cell to another by uptake of apoptotic bodies in a CD4-independent way. This was investigated by coculturing CD4-, chemokine receptor CCR5- and CXCR4- human fetal fibroblasts with apoptotic HIV-1-infected HuT78 cells or apoptotic PBMC isolated from HIV-1-infected patients. After 2 wk of coculture, fibroblasts contained HIV-1 DNA and expressed HIV-1 proteins p24 and gp120. Transfer of HIV-1 DNA was verified by coculturing fibroblasts with apoptotic bodies derived from cells infected with a defective HIV-1 virus. These cells contain one integrated copy of a reverse transcriptase (RT)-negative HIV-1 strain (8E5/LAV RT- cells) and consequently cannot produce free virus. Intracellular HIV-1 gag DNA was detected in both fibroblasts and dendritic cells after coculture with apoptotic 8E5/LAV RT- cells. Transfer of viral DNA after uptake of apoptotic bodies may explain HIV-1 infection of CD4- cells in vivo and furthermore may be relevant for Ag presentation.

Erratum to "Immunocytochemical detection of cytokines and chemokines in Langerhans cells and in vitro derived dendritic cells".

We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14-, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF-alpha, IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1alpha, MIP-1beta and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1alpha and IL-1beta production. IL-1ra and IL-1alpha were expressed in 10-25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1alpha and IL-1beta were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0-5%) synthesized TNF-alpha, IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1alpha, MIP-1beta) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83- and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF-alpha, IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.

Immunocytochemical detection of cytokines and chemokines in Langerhans cells and in vitro derived dendritic cells.

We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14-, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF-alpha, IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1alpha, MIP-1beta and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1alpha and IL-1beta production. IL-1ra and IL-1alpha were expressed in 10-25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1alpha and IL-1beta were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0-5%) synthesized TNF-alpha, IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1alpha, MIP-1beta) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83- and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF-alpha, IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.

T cell subsets in normal human epidermis.

Freshly isolated human lymphocytes from normal epidermis were characterized with respect to distribution of subsets. The major T cell receptor-alpha beta + compartment was enriched for CD4+, for CD8 alpha alpha +, and for CD4-CD8-T lymphocytes compared with peripheral blood lymphocytes. Furthermore, the majority of epidermal T lymphocytes expressed a CD45RA- CD45ROhigh Fas+ memory/effector phenotype; many also expressed early-intermediate activation markers, suggesting antigenic exposure in vivo. The cutaneous lymphocyte-associated antigen was expressed by almost all epidermal T lymphocytes. A large portion also expressed the mucosal-associated alpha e beta 7-integrin, which may mediate retention to epithelium. These data show that T lymphocytes present in normal human epidermis constitute a distinct T cells compartment with characteristics similar to that of other epithelial-associated T cell compartments.

cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I.

Modulation of Tax and PKA-mediated expression of HTLV-I promoter via cAMP response element binding and modulator proteins CREB and CREM.

Nuclear proteins of the human peripheral blood T lymphocytes that bind to the CREs located within three 21-bp repeat enhancers of the HTLV-I promoter belong to the CREB/CREM family of bZIP transcription factors. It has been shown previously that Tax enhances transactivation of these CREs by direct interactions with the bZIP domain of the transcription factors to stabilize DNA-binding. We show that CREB and CREM bind all three CRE sequences of the HTLV-I promoter which are important determinants in Tax-elicited transactivation as well as PKA-mediated activation of the HTLV-I promoter. Tax and PKA activate transcription from a HTLV-I-LTR CAT reporter plasmid transfected to NIH 3T3 cells, and CREM attenuates the activation. In the context of a GAL4 CREB fusion protein in which the DNA-binding bZIP domain of CREB is replaced by GAL4 binding domain, a single amino acid substitution of serine-133, phosphorylated by PKA and critical for the transactivation function of CREB, attenuates both Tax and PKA-mediated transcriptional responses. These observations suggest that Tax enhances CREB-mediated transactivation of the HTLV-I promoter by a mechanism apart from, and/or in addition to, the reported stabilization of DNA-binding by interaction with the bZIP domain of CREB.

No evidence for TCR V beta repertoire changes influencing disease protection in E-transgenic NOD mice.

In order to study whether positive selection of T cells plays any role in the MHC-dependent protection from diabetes in the non-obese-diabetic (NOD) mouse, the T cell V beta repertoire has been studied in NOD mice and in NOD mice either transgenic for the wildtype MHC class II E alpha gene, or for delta Y, a promotor-mutagenized E alpha gene with a restricted tissue expression. The E alpha transgenic line is protected from both insulitis and diabetes. The delta Y transgenic line is neither protected from insulitis nor from diabetes, although it can perform both positive and negative E-mediated selection in the thymus. The V beta repertoire was studied in the pancreatic lymph nodes as these drain the area which is the target for the autoimmune attack. We see no evidence for E alpha TCR V beta repertoire differing from both nontransgenic NOD mice and delta Y mice despite its striking difference in susceptibility to autoimmunity. We conclude that none of the differences in the TCR V beta repertoire of E alpha-transgenic NOD mice hitherto observed are likely to explain the protective effect of E molecule expression in NOD mice.

Co-expression of T-cell receptor beta and delta mRNA detected at high frequency in hybridomas derived from adult thymus.

Two different hybridoma collections from adult C3H/HeJ thymus were generated in order to analyse T-cell receptor (TcR) rearrangements, surface expression of T-cell receptors and differentiation markers as well as lymphokine production. Large, low density thymocytes were either directly fused to the thymoma BW 5147 alpha-beta- variant, or fused after stimulation with Concanavalin A in the presence of interleukin-2 for 48 h. The hybrids obtained from Concanavalin A-stimulated cells represented rather mature thymocytes, with regard to TcR rearrangements and surface T-cell receptor expression. The collection of hybrids derived from freshly isolated large thymocytes contained cells in various stages of T-cell development. An unexpectedly large number of hybrids (46 out of 84) from this group expressed full-length C beta together with full-length, or shorter, C delta mRNA. This finding suggests that a considerable proportion of alpha beta T cells proceeds through a stage in development where delta genes are being rearranged and transcribed.

Induction of CD8 molecules on thymic gamma/delta T cells in vitro is dependent upon alpha/beta T cells.

Thymocytes differentiate upon interactions with microenvironmental components, but the precise role of different stromal cells, or other T cells, in early differentiative events remains unclear. Here we have analyzed the in vitro differentiation of double-negative (DN) thymocytes from young adult mice. We demonstrate that a substantial proportion of DN thymocytes differentiate into CD8+ gamma/delta T cells upon stimulation with concanavalin A and recombinant interleukin 2. However, if alpha/beta T cells are excluded from the initial population of DN thymocytes, the CD8+ gamma/delta T cells do not appear in the cultures. These results suggest a role for T-T cell interactions in thymic differentiative events, and provide evidence for physiological interactions between the alpha/beta and gamma/delta T cell compartments within the thymus.