Conventional protein kinase C plays a critical role in negative regulation of CD98-induced homotypic aggregation.

CD98, a heterodimeric type II transmembrane protein, is involved in many different cellular events, ranging from amino acid transport to cell-cell adhesion. Little is known about the positive and negative signalling pathways involved in these responses. Therefore, we examined the role of conventional protein kinase C (PKC) isoforms during CD98-induced intracellular signalling and homotypic aggregation of U937 cells. The CD98-induced aggregation was enhanced by the general protein kinase inhibitors GF109203X and staurosporin, and by specific PKC-alpha/-beta peptide inhibitor 19-27, but inhibited by PKC activators such as phorbol 12-myristate 13-acetate (PMA). PMA-inhibition was reversed by PKC inhibitors recognising the ATP-binding site in PKC (e.g. staurosporin, GF109203X and Go6983). Inhibitors which bind to diacylglycerol (DAG) or Ca(2+)-binding sites of PKC (calphostin C and Go6967) had no effect. PMA-induced translocation of conventional PKC (cPKC) isozymes (alpha, beta and gamma), but decreased the expression of PKC-delta, which plays an important role in CD98-induced homotypic aggregation. PMA treatment also suppressed the surface level of CD98 but not CD29, CD18 and CD147, dose- and time-dependently. These data provide evidence that PMA-responsive cPKC isoforms (alpha, beta and gamma) play a key role in negative regulation of CD98 signalling and homotypic aggregation.

Autocrine type I interferon amplifies dendritic cell responses to lipopolysaccharide via the nuclear factor-kappaB/p38 pathways.

The central role of dendritic cells (DC) in the initiation of immune responses requires these cells to be able to determine the degree of danger in their microenvironment. Abrogating the activity of type I interferon (IFN) secreted after lipopolysaccharide (LPS) stimulation of DC inhibits CD86 and human leucocyte antigen-DR (HLA-DR) upregulation at a low LPS concentration. At a higher concentration of LPS, while changes in surface phenotype are not dependent on type I IFN, this cytokine is required for maximal secretion of interleukin-12 (IL-12) and tumour necrosis factor-alpha (TNFalpha) by DC. Thus, the secretion and autocrine activity of type I IFN after Toll-like receptor stimulation enables DC to orchestrate a hierarchical maturation response with regard to changes in surface phenotype and secretion of cytokines. In addition, the activation of nuclear factor-kappaB and p38 pathways in DC can occur either in an additive fashion when DC are exposed to dual stimulation or can be activated in discrete phases over time when DC are exposed to LPS alone. The differential activation of these pathways provides a mechanism for DC to integrate the activation by multiple stimuli and thus amplify responses to pathogen infection.

Autologous apoptotic T cells interact with dendritic cells, but do not affect their surface phenotype or their ability to induce recall immune responses.

Dendritic cells (DCs) play an important role in determining immunogenicity and the subsequent immune response. They may also have a role in maintaining peripheral tolerance to self-antigens by initiating an immune response only in the context of danger signals released from cells during stress, damage or death. These signals may originate from surrounding T cells as well as from other cells. Therefore, in this study the effect of autologous T cell injury on DC morphology and function has been investigated. Co-incubation of apoptotic or necrotic T cells with immature DCs altered their morphology towards a more mature appearance, with more cells showing activation as judged by spreading and formation of arborizing long processes. The apoptotic autologous T cells were rarely phagocytosed by immature DCs, compared to macrophages. The DC surface phenotype was not affected by the co-incubation with autologous injured T cells. The ability of DCs to elicit a secondary immune response was not altered by exposure to autologous injured T cells. These findings suggest that DC can continue to function in T cell activation, rather than in tolerogenic mode, even in the presence of large numbers of dying autologous T cells, such as may be present in the aftermath of an acute antiviral response.

Regulation of primary HIV-1 isolate replication in dendritic cells.

The potential role of dendritic cells (DC) in the immunopathology of human immunodeficiency virus 1 (HIV-1) disease remains controversial. This study examines replication of a panel of HIV-1 strains (both laboratory adapted and primary) within DC, in the context of the well-established monocyte-DC and monocyte-macrophage transition. Viral replication was assessed by p24 ELISA assay. All strains of HIV-1 tested replicated in DC. Only CCR5-tropic virus replicated in macrophages. Lipopolysaccharide (LPS) induced DC maturation (as reflected in altered cell phenotype) and at the same time diminished the ability of DC to support HIV-1 replication. In contrast the presence of activated T cells, which had been fixed to prevent them acting as a site for viral replication, enhanced the ability of the DC to support viral replication, as has been reported previously for macrophages. Thus cells that are DC by phenotype, but are not activated, act as the optimum reservoir for HIV-1 replication. If this form of DC is present in peripheral tissues, this will be permissive for amplification of the in vivo viral load at sites where there are few responder cells available, and hence contribute to the persistent immunopathology.

The functional interactions between CD98, beta1-integrins, and CD147 in the induction of U937 homotypic aggregation.

CD98 is expressed on both hematopoietic and nonhematopoietic cells and has been implicated in a variety of different aspects of cell physiology and immunobiology. In this study, the functional interactions between CD98 and other adhesion molecules on the surface of the promonocyte line U937 are examined by means of a quantitative assay of cell aggregation. Several of the CD98 antibodies induced homotypic aggregation of these cells without affecting cellular viability or growth. Aggregation induced by CD98 antibodies could be distinguished from that induced by beta1-integrin (CD29) ligation by lack of sensitivity to EDTA and by increased sensitivity to deoxyglucose. Aggregation induced via CD98 and CD29 could also be distinguished by the pattern of protein tyrosine phosphorylation induced. Some CD29 antibodies partially inhibited CD98-induced aggregation, and these antibodies were neither agonistic for aggregation nor inhibitors of beta1-integrin binding to substrates. Conversely, some CD98 antibodies were potent inhibitors of CD29-induced aggregation. Antibodies to beta2 integrins also partially inhibited CD98-induced aggregation. Unexpectedly, 2 antibodies to CD147, an immunoglobulin superfamily member whose function has remained unclear, were also potent inhibitors of both the aggregation and the protein tyrosine phosphorylation induced via CD98 ligation. The results of this study support a central role for CD98 within a multimolecular unit that regulates cell aggregation.

Nonradioactive detection of the common Connexin 26 167delT and 35delG mutations and frequencies among Ashkenazi Jews.

Mutations in the gap junction beta2 (GJB2) gene, Connexin 26 (Cx26), cause nonsyndromic sensorineural recessive deafness (NSRD). Two frameshift mutations, 167delT and 35delG, are the most frequent Cx26 lesions causing NSRD. The 35delG mutation is panethnic, while the 167delT lesion occurs almost exclusively in the Ashkenazi Jewish population at a carrier frequency of 2 to 4%. To facilitate carrier detection, a simple nonradioactive allele-specific oligonucleotide (ASO) hybridization assay was developed for the 167delT and 35delG mutations. Screening of 1012 anonymous Ashkenazi Jewish individuals from the New York Metropolitan area revealed carrier frequencies for 167delT and 35delG of 3.96% (95% CI: 2.75-5.15%) and 0.69% (95% CI: 0.18-1.20%), respectively. This sensitive, specific, and relatively inexpensive method can reliably identify affected newborns and patients with NSRD as well as facilitate carrier screening for Connexin 26 deafness in the Ashkenazi Jewish community.

Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria.

The early role of natural killer cells and gamma delta T cells in the development of protective immunity to the blood stage of nonlethal Plasmodium yoelii infection was studied. Splenic cytokine levels were measured 24 h after infection of natural killer cell-depleted immunodeficient and littermate mice or transiently T-cell-depleted normal mice. Splenic gamma interferon levels were significantly increased above background in immunodeficient and littermate mice 24 h after infection. Depletion of natural killer cells resulted in markedly depressed gamma interferon levels and poor control of parasitemia, particularly in severe combined immunodeficient mice. In the littermates, gamma interferon levels were partially reduced, but parasitemias were resolved normally. However, in athymic mice, natural killer cell depletion had no effect on gamma interferon production. Levels of tumor necrosis factor alpha were increased in all animals 24 h after infection, and responses were not affected by natural killer cell depletion. However, in T-cell-depleted animals, both gamma interferon and tumor necrosis factor alpha levels were decreased 24 h after infection, and depleted mice were unable to control their parasitemia. These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and gamma delta T cells contribute equally towards their production. The data also suggest that the subsequent resolution of infection requires early production of gamma interferon, which might act by switching on the appropriate T-helper-cell subsets and other essential parasitotoxic effector mechanisms.

Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature.

A murine model that closely resembles human cerebral malaria is presented, in which characteristic features of parasite sequestration and inflammation in the brain are clearly demonstrable. "Young" (BALB/c x C57BL/6)F(1) mice infected with Plasmodium berghei (ANKA) developed typical neurological symptoms 7 to 8 days later and then died, although their parasitemias were below 20%. Older animals were less susceptible. Immunohistopathology and ultrastructure demonstrated that neurological symptoms were associated with sequestration of both parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions. Increases in tumor necrosis factor alpha and CD54 expression were also present. Similar phenomena were absent or substantially reduced in older infected but asymptomatic animals. These findings suggest that this murine model is suitable both for determining precise pathogenetic features of the cerebral form of the disease and for evaluating circumventive interventions.

Self/nonself discrimination among immunoregulatory (CD4) T cells.

This review covers work on immunological tolerance from 1962 up to the present, focusing on the Th, CD4+ compartment of the immune system. The principle mechanism of tolerance is identified as deletion, occurring centrally and in the periphery. In the periphery, deletion is the normal response of CD4 T cells to soluble monomeric proteins that occurs when activation (mainly of dendritic cells) is avoided. Thus activation and the signals which induce it are crucial to understanding S/NS discrimination, as has long been known. The thymus is important as the site where new T cells first see self-antigens, and as one largely shielded from activation, although deletion in the thymus and the periphery has the same threshold. The relative contribution of dendritic cells and developing T cells to deletion in the thymus remains unclear. Activation induced cell death, containment, anergy and deviation constitute subsidiary mechanisms, and sequestration/neglect is important in limiting the scope of deletion.

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Wiskott-Aldrich syndrome protein is necessary for efficient IgG-mediated phagocytosis.

Interactions between the Wiskott-Aldrich (WAS) protein (WASp), small GTPases, and the cytoskeletal organizing complex Arp2/3 appear to be critical for the transduction of signals from the cell membrane to the actin cytoskeleton in hematopoietic cells. This study shows that Fcgamma-receptor (FcgammaR)-mediated phagocytosis is impaired in WASp-deficient peripheral blood monocytes, and that in macrophages, formation of the actin cup and local recruitment of tyrosine phosphorylated proteins is markedly attenuated. Results also show that, in normal macrophages, WASp itself is actively recruited to the cup, suggesting that assembly of this specialized cytoskeletal structure is dependent on its expression. (Blood. 2000;95:2943-2946)

Expression of retinoid receptors during human monocyte differentiation in vitro.

1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)VD(3)) and retinoic acid (RA) modulate the activation of monocytes (MO) and their differentiation into macrophages (MAC). As these effects are mostly mediated by heterodimers or homodimers of the specific nuclear receptors for 1,25(OH)(2)VD(3) and RA, we investigated the expression of the retinoic acid receptors (RAR) alpha, beta, and gamma and the retinoid X-receptor (RXR) alpha in MO during differentiation into MAC or dendritic cells (DC). The mRNA of all investigated receptors except RARbeta was detected in short-term cultured MO. During differentiation of MO to MAC the mRNA expression of the RA receptors decreased. In contrast, along the differentiation pathway of MO to DC, only the mRNA expression of RARgamma declined, whereas RARalpha and RXRalpha were constantly expressed at a high level. Despite the strong expression of RARalpha and RXRalpha at mRNA level in MO-derived DC, the protein expression of the receptors was low in these cells. However, MO and MO-derived MAC showed a strong expression of these receptors at protein level. This suggests that a posttranscriptional or posttranslational mechanism of receptor regulation is occurring in these cells, and in particular in the DC. The inverse regulation of RA receptor expression and protein levels between MAC and DC may control the responsiveness of these cells to 1,25(OH)(2)VD(3) and RA.

Acute intermittent porphyria: novel missense mutations in the human hydroxymethylbilane synthase gene.

PURPOSE: To identify mutations in families with acute intermittent porphyria, an autosomal dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase. METHODS: Mutations were identified by direct solid phase sequencing. RESULTS: Two novel missense mutations E80G and T78P and three previously reported mutations, R173W, G111R, and the splice site lesion, IVS1+1, were detected, each in an unrelated proband. The causality of the novel missense mutations was demonstrated by expression studies. CONCLUSION: These findings provide for the precise diagnosis of carriers in these families and further expand the molecular heterogeneity of AIP.

Vectorial function of major histocompatibility complex class II in a human intestinal cell line.

This study explores the expression and the function of major histocompatibility complex class II in the intestinal epithelial cell line CaCo-2, which has been widely used as a model for the human gastrointestinal epithelium. Human leucocyte antigen (HLA)-DR expression on CaCo-2 cells is induceable by interferon-gamma (IFN-gamma), but responsiveness to IFN-gamma is dependent on cell differentiation and IFN-gamma availability at the basolateral cell surface. HLA-DR expression is concentrated in apical cytoplasmic vesicles and on the basolateral cell surface. Invariant chain is expressed in apical vesicles but is absent from the cell surface. Immunoprecipitation studies show a slow rate of dissociation of HLA-DR from Ii. Double labelling shows some overlap between HLA-DR expression and basolateral endosomal markers but no overlap with apical endosomal markers. Functional studies show processing and presentation of lysozyme endocytosed from the basolateral, but not apical surfaces. CaCo-2 cells may provide a useful model with which to dissect the antigen-processing pathways in polarized epithelial cells. The regulated access of antigens taken up from the gut lumen to the processing compartments may prevent overloading the immune system with antigens derived from normal gut contents.

Myocyte loss in chronic heart failure.

This study examined whether or not there is progressive loss of individual myocytes in established heart failure, accounting for the progressive left ventricular dysfunction; whether such loss is by necrosis or apoptosis; and whether such loss is more pronounced in ischaemic heart disease or idiopathic dilated cardiomyopathy. Tissue for patients undergoing cardiac transplantation for clinical end-stage heart disease was used. The clinical diagnosis was not known to the observer at the time of analysis. Indices of potential myocyte loss were: detection of apoptotic nuclei in situ, using the TUNEL method, immunohistochemistry for CD120a, CD120b, CD95, perforin and granzyme B; binding of C9 complex; and lipofuscin deposition within macrophages. Interstitial macrophages and T cells and their relationship to myocyte loss were also examined. There is indeed low grade myocyte loss in chronic heart failure, but there was no difference between the disease groups; rather, there was marked patient-to-patient variation within each category. Thus in chronic heart failure myocyte loss does occur, and both necrosis and apoptosis contribute to this loss, irrespective of the underlying nature of the disease. Any mechanism which accounts for myocyte loss must be common to both conditions, rather than specific for a pre-operative diagnosis.

Molecular characterization of U937-dependent T-cell co-stimulation.

U937 cells provide a co-stimulatory signal for CD3-mediated T-cell activation which is independent of the CD28/CD80/CD86 interaction. This study set out to identify which molecules contribute to this co-stimulatory activity. Monoclonal antibodies (mAb) to the known accessory molecules CD11a, CD18, CD54 and CD45, all inhibited T-cell proliferation. Although CD11a/18 mAb inhibited U937/T-cell cluster formation as well as proliferation, CD45 enhanced the size of the clusters formed, suggesting that this was not the only mechanism of inhibition. The alternative co-stimulatory pathway provided by U937 cells preferentially stimulated a response in the CD18+ T-cell population, and this reflected the reduced sensitivity of CD8+ T cells to CD28-mediated activation. Monoclonal antibodies to three molecules, CD53, CD98 and CD147, also inhibited U937-dependent T-cell proliferation. The mAb to CD98 and CD147 were inhibitory when prepulsed on to the U937 cells, suggesting an effect mediated by these molecules on the antigen-presenting cell.

Reactive oxygen species activate human peripheral blood dendritic cells.

This study investigates the effects of hydrogen peroxide, a potent oxygen free radical donor, on the phenotype and function of dendritic cells differentiated from peripheral blood precursors. We report that hydrogen peroxide induces an up-regulation of several dendritic cell surface markers involved in interaction with T cells, including MHC Class II molecules (DQ and DR) and the co-stimulatory molecules CD40 and CD86. Moreover we have observed that H2O2-treated dendritic cells are more efficient in promoting T cell proliferation than normal dendritic cells and that this enhancement can be blocked using the free radical scavenger agent N-acetylcysteine. Oxygen free radicals are a common by-product of inflammation, and our results suggest they may play an important role in activation of sentinel dendritic cells, linking tissue damage to the initiation of an adaptive immune response.

Responses to human rhinovirus in CD45 T cell subsets isolated from tonsil.

Isotypes of CD45 have been used extensively as markers of memory and naive populations of T cells in peripheral blood. In this study, T cells were isolated from human tonsil and their proliferative response against human rhinovirus was measured. Unexpectedly, equivalent responses were found among the CD4+CD45RA+ and CD4+CD45RO+ populations of T cells. This response requires MHC class II-positive antigen-presenting cells. The time course of the T cell response in vitro was that of a classical recall response, and no proliferative response to the virus could be detected in human cord blood. These results suggest that tonsils contain a significant population of CD45RA+ memory cells. The presence of this population may reflect ongoing stimulation with this common infectious agent, and the anatomical location of the T cells within the major lymphoid organ draining the naso-pharyngeal epithelial surface.