Expression Of Beta Chain Research Articles

Alterations in CIITA constitute a common mechanism accounting for downregulation of MHC class II expression in diffuse large B-cell lymphoma (DLBCL)

Significant decreases in patient survival are associated with downregulation of major histocompatibility complex class II (MHC-II) antigen expression in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms responsible for decreased MHC-II expression in DLBCL are poorly defined. We therefore examined these mechanisms in established DLBCL cell lines. Human leukocyte antigen (HLA)-DR surface expression was examined by flow cytometry. Expression of the MHC-II genes and the MHC-II transcriptional activators class II transactivator (CIITA) and RFX was investigated by reverse transcriptase polymerase chain reaction. The integrity of the MHC-II genes was examined by polymerase chain reaction. Stable transfection assays were utilized to reconstitute CIITA expression. Dramatic variations in the levels of cell surface HLA-DR expression were observed on the DLBCL cell lines. OCI-Ly10 cells lack HLA-DR and HLA-DQ expression due to homozygous deletions within the MHC-II locus on chromosome 6. Dyscoordinate downregulation of MHC-II beta-chain expression in OCI-Ly3 cells mediates dramatic reductions of MHC-II surface expression. In SUDHL-4 and SUDHL-6 cells, expression of the MHC-II genes is coordinately reduced and quantitatively correlated with expression of the CIITA, the master regulator of MHC-II transcription. DB cells lack expression of CIITA and all of the MHC-II genes. Stable transfection of DB cells with CIITA expression vectors resulted in coordinate upregulation of MHC-II gene expression, which demonstrates the causal relationship between the lack of CIITA and MHC-II loss. These data demonstrate that downregulation of MHC-II expression occurs by multiple distinct mechanisms in DLBCL. However, decreases in CIITA expression appear to be the most prevalent mechanism.

Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells

Although digitalis has been used in clinical treatment extensively, the precise mechanism of its toxic actions on cardiovascular system remained unclear, it would be of interest to study the differential proteomic analysis of vascular endothelial cells in response to toxic concentrations of digitalis thus to provide new agents for treatment of digitalis-induced cytotoxicity. We employed human umbilical vein endothelial cells (HUVEC) as our model system. HUVEC were exposed to increasing concentrations (0.1 nM–10 μM) of digoxin at 12–96 h intervals. Cell viability tests revealed that digoxin played dual effects on cell growth. Apoptosis detection confirmed that apoptosis was primarily responsible for digoxin-induced cell death. Proteomics analysis further revealed that the digoxin-induced apoptosis was accompanied by regulated expression of ATP synthase beta chain, cystatin A, electron transfer flavoprotein, heterogeneous nuclear ribonucleoproteins H3, lamin A, profilin-1, proteasome subunit 5, succinyl-CoA ligase beta chain and heat shock protein 60 (HSP60). Deep study on the overexpression of HSP60 confirmed that HSP60 exerted a protective role in digoxin-induced apoptosis through inhibition of caspase-3 activity in HUVEC. These results provided an impetus for further delineation of mechanism of digoxin-induced cytotoxicity and offered new agents that help attenuate its toxicity.

Nicotinic acid: recent developments

To review the recent progress in niacin research that is made in two major areas: new preparations to decrease flushing and niacin's mechanism of action. Flushing, an adverse effect of niacin, results from GPR109A-mediated production of prostaglandin D2 and E2 in Langerhans' cells which act on DP1 and EP2/4 receptors in dermal capillaries causing their vasodilatation. DP1 receptor antagonist (laropiprant) attenuates the niacin flush in animals and humans. A reformulated preparation of extended-release niacin lowers flushing compared with the extended-release niacin (Niaspan, Abbott Laboratories, Chicago, Illinois, USA). Aspirin pretreatment attenuates flushing from Niaspan. Recent data on niacin's mechanism of action indicate that it directly inhibits hepatic diacylglycerolacyl transferase 2 resulting in an inhibition of triglyceride synthesis and decreased apolipoprotein B-containing lipoproteins; niacin, by inhibiting the surface expression of hepatic ATP synthase beta chain, decreases the hepatic holoparticle high-density lipoprotein catabolism and raises high-density lipoprotein levels; and niacin increases redox potential in arterial endothelial cells resulting in the inhibition of redox-sensitive genes. Recent developments suggest that the niacin receptor GPR109A is involved in flushing, but it does not explain multiple actions of niacin. Actions of niacin on diacylglycerolacyl transferase 2, ATP synthase beta chain, and redox state may explain the multiple actions of niacin.

Study of IL-2 receptor expression after chemoimmunotherapy in patients treated for metastatic malignant melanoma.

Using flow cytometry, cellular IL-2 receptors were studied before and following chemoimmunotherapy combination in 20 patients with metastatic malignant melanoma (MMM). Patients received cisplatin (100 mg/m2) at days 1 and 28, recombinant IL-2 by continuous infusion from days 3 to 6, 17 to 21, 31 to 34, and 45 to 49. Interferon-alpha (IFN-alpha) was given subcutaneously three times weekly. In terms of clinical response, we observed 55% objective response (complete: 15%). When pretreatment blood samples were compared with those of healthy donors, we did not observe any change in low (alpha chain) and high affinity receptor (alpha + beta) expression. In contrast, intermediate affinity p75 (beta chain) expression was decreased significantly (P < or = 0.0001) in MMM patients. During treatment, we found a dramatic increase of beta chain as well as high affinity (alpha + beta) expression in responding patients, as soon as IL-2 therapy began. Furthermore, the increase of beta chain expression was limited to natural killer (NK) cells (CD56+). In non-responding patients, on the other hand, increase of both receptors was seen only at day 31. These data suggest the involvement of beta chain expression in the mechanism of cell activation after chemoimmunotherapy. Moreover, this early beta chain expression is correlated with the clinical response to chemoimmunotherapy.

Nicotinic Acid (Niacin) Receptor Agonists: Will They Be Useful Therapeutic Agents?

Nicotinic acid (niacin) favorably affects very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and lipoprotein (a) (LP[a]) and increases high-density lipoprotein (HDL). Emerging data indicates vascular anti-inflammatory properties to additionally account for niacin's proven effects in cardiovascular disease. Recent evidence indicates that niacin acts on GPR109A and GPR109B (HM74A and HM74, respectively), receptors expressed in adipocytes and immune cells. In adipocytes, GPR109A activation reduces triglyceride (TG) lipolysis, resulting in decreased free fatty acid (FFA) mobilization to the liver. In humans, this mechanism has yet to be confirmed because the plasma FFA decrease is transient and is followed by a rebound increase in FFA levels. New evidence indicates niacin directly inhibits diacylglycerol acyltransferase 2 (DGAT2) isolated from human hepatocytes, resulting in accelerated hepatic apolipoprotein (apo)B degradation and decreased apoB secretion, thus explaining reductions in VLDL and LDL. This raises important questions as to whether stimulation of GPR109A in adipocytes or inhibition of DGAT2 in liver by niacin best explain the reduction in VLDL and LDL in dyslipidemic patients. Kinetic and in vitro studies indicate that niacin retards the hepatic catabolism of apoA-I but not liver scavenger receptor B1-mediated cholesterol esters, suggesting that niacin inhibits hepatic holoparticle HDL removal. Indeed, recent preliminary evidence suggests that niacin decreases surface expression of hepatic beta-chain of adenosine triphosphate synthase, which has been implicated in apoA-I/HDL holoparticle catabolism. GPR109A-mediated production of prostaglandin D2 in macrophages and Langerhan cells causes skin capillary vasodilation and explains, in part, niacin's effect on flushing. Development of niacin receptor agonists would, theoretically, result in adipocyte TG accumulation (and clinical adiposity) and increased flushing. This raises questions about niacin receptor agonists as therapeutic agents. Several niacin receptor agonists have been developed and patented, but their clinical effects have not been described. Future research is needed to determine whether niacin receptor agonists will demonstrate all the beneficial properties of nicotinic acid on atherosclerosis and without significant adverse effects.

SIMVASTATIN REDUCES INSULIN-LIKE GROWTH FACTOR-1 SIGNALING IN DIFFERENTIATING C2C12 MOUSE MYOBLAST CELLS IN AN HMG-CoA REDUCTASE INHIBITION-INDEPENDENT MANNER

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase occasionally cause myopathy characterized by weakness, pain, and elevated serum creatine phosphokinase (CK). In this study, we investigated the effects of simvastatin, an HMG-CoA reductase inhibitor, on the viability and insulin-like growth factor-1 (IGF-1) signaling in differentiating C2C12 mouse myoblast cells. Simvastatin decreased cell viability and CK activity, a marker of myogenesis, in differentiating cells in a dose-dependent manner. Although the simvastatin-induced decrease in viability in proliferating and differentiated cells was completely abolished by mevalonate or geranylgeranyl-pyrophosphate, the inhibitory effects of simvastatin in differentiating cells were not abolished by mevalonate or isoprenoid derivatives of mevalonate. Moreover, the sensitivity of differentiating cells to simvastatin regarding cell viability was about 7 times higher than that of proliferating cells. After induction of differentiation in the presence of 1 microM simvastatin for 2 days, IGF-1-induced activation of ERK1/2 and Akt was significantly decreased. Although mRNA expression of the IGF-1 receptor beta-chain (IGF-1R beta) did not change, protein level of the 200 kDa IGF-1Rbeta precursor was significantly increased by simvastatin in a dose-dependent manner. Mevalonate did not abolish the effect of simvastatin on IGF-1Rbeta expression. These results suggest that simvastatin decreases IGF-1 signaling via a regulation of the post-translational modification of IGF-1Rbeta in an HMG-CoA reductase inhibition-independent manner.

Celecoxib Inhibits Interleukin-12 αβ and β2Folding and Secretion by a Novel COX2-Independent Mechanism Involving Chaperones of the Endoplasmic Reticulum

Celecoxib (CE) is a nonsteroidal anti-inflammatory drug (NSAID) that is a specific inhibitor of cyclooxygenase 2 (COX2). It is indicated for a variety of chronic inflammatory conditions, including rheumatoid arthritis. Over the last few years, adverse cardiovascular effects and increased risk for heart attacks have been associated with this drug. In addition, evidence is emerging for COX2-independent molecular targets. CE has been shown to induce apoptosis in various cancer cells lines through a COX2-independent mechanism that seems to involve inactivation of protein kinase Akt and inhibition of endoplasmic reticulum (ER) Ca2+ ATPase. In this study, we show that both CE and an analog devoid of COX2 inhibitory activity [1-(4-sulfamoyl phenyl)-3-trifluoromethyl-5-(4-trifluoromethylphenyl)pyrazole, CEA] inhibit the secretion of the dimeric interleukin-12 (IL-12) alphabeta and beta2 forms with identical IC50 values of 20 and 30 microM, respectively, whereas no such effect was seen with rofecoxib. Reverse transcription-polymerase chain reaction analysis showed that this inhibition was not due to a blockage of transcription of the alpha- and beta-chain expression cassettes. Secretion of the beta monomer form was less strongly inhibited, suggestive for a mechanism primarily targeting dimer assembly in the ER. Analysis of intracellular fractions revealed that both CE and CEA increased the association of IL-12 with calreticulin, an endoplasmic reticulum-resident chaperone involved in the retention of misfolded cargo proteins while blocking interaction with ERp44. Our findings reveal a previously undescribed effect of celecoxib on oligomer protein folding and assembly in the endoplasmic reticulum and ensuing secretion and suggest that celecoxib-driven alteration of the secretome may be involved in some of its clinical side effects.

GATA-1 is required for expression of FcεRI on mast cells: analysis of mast cells derived from GATA-1 knockdown mouse bone marrow

The high-affinity receptor for IgE (FcepsilonRI) that is expressed on the surface of mast cells plays an important role in antigen/IgE-mediated allergic reactions. We have previously found that critical elements in the promoter of the FcepsilonRI alpha- and beta-chain genes are recognized by the transcription factor GATA-1 in electrophoretic mobility shift assays coupled with a transient expression system for the alpha- and beta-chain promoters. To confirm that GATA-1 is involved in the expression of FcepsilonRI definitively, we generated bone marrow-derived mast cells from GATA-1 knockdown (KD) heterozygous mice. FACS analysis showed that the frequency of FcepsilonRI-positive cells was significantly decreased in mast cells derived from bone marrow of GATA-1 KD mice. Reverse transcription-PCR analysis showed that the level of transcripts not only for GATA-1 but also for both the alpha- and beta-chains was significantly lower in KD mast cells, whereas that of the FcepsilonRI gamma-chain was not affected. Degranulation caused by cross-linking of FcepsilonRI on mast cells prepared from KD mice was markedly repressed in comparison with that of wild-type mast cells. We concluded that the transcription factor GATA-1 positively regulates FcepsilonRI alpha- and beta-chain expression and therefore is involved in mast cell development.

Rosiglitazone produces insulin sensitisation by increasing expression of the insulin receptor and its tyrosine kinase activity in brown adipocytes.

Rosiglitazone is used to treat Type 2 diabetes because it improves insulin sensitivity. However, the specific molecular mechanism by which this compound acts has not yet been explained. We used fetal rat primary brown adipocytes cultured for 24 h with or without 10 micro mol/l rosiglitazone and further stimulated for 5 min with 10 nmol/l insulin. Next we measured glucose uptake and GLUT4 translocation and submitted the cells to lysis, immunoprecipitation and immunoblotting in order to measure the insulin signalling cascade. Rosiglitazone noticeably activated basal glucose uptake in a manner dependent on p38-mitogen-activated protein kinase. Rosiglitazone also produced a 40% increase in insulin-stimulated glucose uptake as a result of increased GLUT4 translocation to the plasma membrane. This happened without changes in the expression of GLUT4 at the mRNA or protein level. This effect correlated with the potentiation by rosiglitazone of insulin-stimulated Tyr phosphorylation of insulin receptor substrate-1 and to a greater extent of insulin receptor substrate-2. It also correlated with the subsequent activation of phosphatidylinositol 3-kinase and Akt, without changes in protein kinase Czeta activity. Rosiglitazone treatment increased insulin receptor expression and insulin-stimulated Tyr phosphorylation of insulin receptor beta-chain, but decreased insulin-stimulated Ser phosphorylation. It also potentiated insulin-induced Tyr phosphorylation of insulin receptor beta-chain and protein tyrosine phosphatase 1B in co-immunoprecipitates and impaired insulin activation of protein tyrosine phosphatase 1B activity. At the insulin receptor level, rosiglitazone-induced improvements of insulin sensitivity result from two convergent mechanisms: increased insulin receptor expression and insulin receptor activation.

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis and in vitro cytokine-stimulated blood eosinophils.

Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha and beta chain and interleukin (IL)-3 receptor alpha chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor beta chain and IL-3 receptor alpha chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-gamma and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-gamma. The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.

The function role of GATA-3 in Th1 and Th2 differentiation.

GATA-3 plays a central role in regulating Th1 and Th2 cell differentiation. Upon interleukin (IL)-4 binding to its receptor, GATA-3 is induced through the action of Stat6. GATA-3 regulates Th2 cytokine expression not only at the transcription level, such as directly binding to the promoters of the IL-5 and IL-13 gene, but also by the involvement in the remodeling of the chromatin structure and opening the IL-4 locus. As a master control, GATA-3 stabilizes the Th2 phenotype by two methods. First, GATA-3 shuts down Th1 development through the repression the IL-12 receptor beta2-chain expression. Second, GATA-3 augments its own expression by a positive feedback autoregulation. In this article, we review the recent study of the function of GATA-3 in Th1 and Th2 differentiation.

Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's β-amyloid 1–42

It has been demonstrated that immunization of transgenic mouse models of Alzheimer's disease (AD) with amyloid-β 1–42 peptide (Aβ 1–42) results in prevention of Aβ plaque formation and amelioration of established plaques in the brain. As the response of the T lymphocyte helper (Th) arm of the immune response had not yet been investigated after Aβ immunization, we i.p. immunized C57BL/6 mice with Aβ 1–42, Aβ 1–40, or phosphate-buffered saline (PBS), and examined markers of Th1 and Th2 immune responses in spleen and in splenocytes from these mice. Spleens from Aβ 1–42-immunized mice demonstrated decreased interleukin-12 receptor beta chain expression compared to mice immunized with Aβ 1–40 or PBS. Consistently, following stimulation with concanavalin A or anti-CD3 antibody, primary splenocytes from Aβ 1–42-immunized mice demonstrated elevated secretion of interleukin-4 and interleukin-10, and decreased levels of interferon-γ. To validate this Th1→Th2 shift in a transgenic mouse model of AD, we immunized Tg APP sw mice (line 2576) with Aβ 1–42 and found decreased Th1 (interleukin-2 and interferon-γ) and elevated Th2 (interleukin-4 and interleukin-10) cytokines in their stimulated primary splenocytes. Interferon-γ was markedly reduced and interleukin-10 was increased in blood plasma from these mice, effects that were associated with dramatically mitigated Aβ deposition after Aβ 1–42 immunization. Taken together, these results show enhanced Th2 and down-regulated Th1 immunity following immune challenge with Aβ 1–42.

Synergistic inhibitory activities of interleukin-10 and dexamethasone on human CD4+ T cells.

We have previously demonstrated that interleukin (IL)-10 synergizes with dexamethasone (Dex) in inhibiting proliferation of human T cells, stimulated in an antigen-presenting cell (APC)-dependent manner. Because IL-10 effectively inhibits APC accessory functions, the synergism could have been a result of its effect on APC. We then investigated the effects of Dex and IL-10 on T-cell subpopulations, stimulated in an APC-independent manner. CD4 and CD8 T cells were stimulated with anti-CD3, with or without Dex and IL-10, alone or in combination. Proliferation, glucocorticoid (GC) receptor binding, anti-CD3-induced tyrosine phosphorylation, IL-2 production, and expression of IL-2 receptor alpha, beta, and gamma chains were evaluated. The pharmacologic interactions were analyzed using the isobole method. IL-10 synergized with Dex in inhibiting CD4 but not CD8 T-cell proliferation. The synergism was not associated with modifications of GC receptor number or affinity, nor with modifications of anti-CD3-induced tyrosine phosphorylation. IL-10 synergized with Dex in inhibiting IL-2 production and increased Dex inhibitory effect on the expression of the IL-2 receptor alpha chain, which is up-regulated by CD3 stimulation and IL-2. Only Dex inhibited the beta and gamma chain expression, which, interestingly, is not up-regulated by IL-2. IL-2, as well as IL-7 and IL-15, reversed the effects of IL-10 but not those of Dex. IL-10 synergizes with Dex in inhibiting CD4 T-cell proliferation. Its synergizing effect is mediated by the inhibition of IL-2 production. Dex exerts additional activities, such as the inhibition of beta and gamma chain expression. Therefore, IL-10 could be useful for the enhancement of GC-based immunosuppressive therapies.

T(h)1/T(h)2 cell differentiation of developing CD4 single-positive thymocytes.

In this study we investigate the stage at which developing T cells in the thymus acquire the ability to differentiate into T(h)1 and T(h)2 cells. We addressed this question by using sorted heat-stable antigen (HSA)(+) and HSA(-) CD4 single-positive (SP) thymocytes prepared from ovalbumin-specific TCRalphabeta transgenic mice and an in vitro T(h)1/T(h)2 differentiation culture system. HSA(-) CD4 SP thymocytes show nearly full functional capacity to differentiate into either T(h)1 or T(h)2 cells. A dramatic difference was observed, however, between HSA(+) and HSA(-) CD4 SP thymocytes in the efficiency for T(h)1 cell differentiation. TCR function of HSA(+) CD4 SP thymocytes appeared to be fully developed because antigen-induced proliferation and IL-2 production were essentially equivalent to that of HSA(-) CD4 SP thymocytes. However, the levels in IL-12 receptor (IL-12R) beta2 chain expression following anti-TCR stimulation were dramatically low in the HSA(+) CD4 SP thymocytes. Decreased IL-12-induced STAT4 phosphorylation was also observed. Moreover, IL-12-dependent transcriptional up-regulation of T-bet and STAT4 was deficient in the HSA(+) CD4 SP thymocytes. Thus, the poor capacity of HSA(+) CD4 SP thymocytes to proceed to T(h)1 cell differentiation appears to be at least partly due to underdeveloped capacity in IL-12R expression and function.

Expression of interleukin-18 receptor in fibroblast-like synoviocytes.

An excess of the proinflammatory substance IL-18 is present in joints of patients with rheumatoid arthritis (RA), and expression of IL-18 receptor (IL-18R) regulates IL-18 bioactivity in various cell types. We examined the expression of IL-18R alpha-chain and beta-chain and the biologic effects of IL-18 in fibroblast-like synoviocytes (FLS) after long-term culture. The presence of both IL-18R chains was a prerequisite for IL-18 signal transduction in FLS. However, all FLS cultures studied were either resistant or barely responsive to IL-18 stimulation as regards cell proliferation, expression of adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, and the release of interstitial collagenase and stromelysin, IL-6 and IL-8, prostaglandin E2, or nitric oxide. We conclude that the presence of macrophages or IL-18R+ T cells that can respond directly to IL-18 is essential for the proinflammatory effects of IL-18 in synovitis in RA.

Dysregulation of Lymphocyte Interleukin-12 Receptor Expression in Sézary Syndrome

Initial phase I and II clinical trials with recombinant human interleukin-12 have demonstrated the therapeutic efficacy of this cytokine in early stage cutaneous T cell lymphoma as compared with more advanced stages such as the leukemic Sézary syndrome. In an effort to optimize the use of recombinant human interleukin-12, using flow cytometry we studied the regulation of the interleukin-12 receptor beta1 (high affinity chain) and beta2 (chain necessary for interleukin-12 signal transduction) on normal volunteer CD4+ and CD8+ T cells and CD4+ and CD8+ cells from eight patients with different degrees of leukemic involvement with Sézary syndrome. The beta1 chain was not readily detectable on resting normal and T cells from Sézary patients, but expression was induced following T cell activation with phytohemagglutinin. Similarly, the beta2 chain was not detectable on resting normal volunteer T cells, but could be induced following phytohemagglutinin stimulation. Moreover, the beta2 chain on normal volunteer T cells was markedly upregulated following short-term culture with interferon-gamma or recombinant human interleukin-12. CD8+ T cells routinely exhibited a greater expression of beta2 than did CD4+ T cells. In marked contrast, both CD4+ and CD8+ T cells from patients with Sézary syndrome and a high tumor cell burden (> 50% circulating atypical Sézary T cells) failed to express the beta2 chain under any culture conditions. Although, culture with anti-interleukin-10 also markedly increased beta2 expression on normal volunteer T cells, this failed to induce expression on either CD4+ or CD8+ T cells from Sézary patients and a high tumor burden. Investigation of patients with Sézary syndrome and a low tumor cell burden (< 15% circulating Sézary T cells) revealed a pattern of beta2 expression that was intermediate between advanced Sézary syndrome and normal volunteers. Both CD4+ and CD8+ peripheral blood T cells from these earlier stage patients were induced to express the beta2 chain, although at a lower frequency of positivity than T cells from normals, following culture with phytohemagglutinin, interferon-gamma, recombinant human interleukin-12, or anti-interleukin-10. These results indicate that short-term culture with interferon-gamma and recombinant human interleukin-12 potently upregulates beta2 chain expression on T cells from normal volunteers, whereas a similar, but less marked effect occurs on T cells from Sézary syndrome patients and a low circulating tumor cell burden. In contrast, the beta2 chain appears to be suppressed on both CD4+ and CD8+ T cells from Sézary patients with a heavy circulating tumor cell burden and it is not induced by interferon-gamma or recombinant human interleukin-12. Therefore, recombinant human interleukin-12 is likely to be most effective for early stage cutaneous T cell lymphoma due to a greater display of beta2 receptors on responding CD8+ anti-tumor cytotoxic T cells.

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts.

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).

The effect of TGF-beta1 on immune responses of naïve versus memory CD4+ Th1/Th2 T cells.

The role of TGF-beta1 in the regulation of T cell responses has been perplexing, possibly because it is dependent on the type of T cell being regulated and its cytokine microenvironment. In the present study, we demonstrate that TGF-beta1 has a profound inhibitory effect on naive CD4+ T cell undergoing differentiation under defined neutral, Th1 and Th2 priming conditions. In addition, we show that if CD4+ T cells are primed in the presence of TGF-beta1, they exhibit reduced secondary anti-CD3/anti-CD28-induced and antigen-specific immune responses (even when TGF-beta is absent during the secondary response), which is not due to reduced expression of co-stimulatory molecules or to inadequate IL-2 production. Finally, with respect to the effect of TGF-beta on fully differentiated antigen-specific memory CD4+ T cells, we demonstrate that while antigen-specific activation and cytokine secretion by memory Th1 T cells is inhibited by TGF-beta1, such inhibition is associated with partial down-regulation of IL-12 receptor beta2 chain expression. In contrast, memory Th2 T cells are not subject to TGF-beta1 -mediated suppression. In summary, these studies reveal that TGF-beta1 is a powerful negative regulator of the primary immune response of CD4+ T cells, but only Th1 T cells are subject to such regulation after the memory stage of T cell differentiation has been reached. Thus, these studies define the potential regulatory role of TGF-beta1 in Th1 and Th2 T cell-mediated autoimmunity.

Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors.

The importance of IL-2Rbeta function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rbeta-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rbeta function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R beta-chain in IL-2Rbeta-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Ralpha expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rbeta provides an essential signal during thymic development to regulate self-reactivity.

Progressive expansion of an L-selectin-negative CD8 cell with anti-feline immunodeficiency virus (FIV) suppressor function in the circulation of FIV-infected cats.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by the appearance of a major CD8 subpopulation with reduced expression of the CD8 beta chain (CD8alpha+betalo). CD8 antiviral activity was subsequently shown to be mediated by the CD8alpha+betalo phenotype, which is the dominant CD8 phenotype in long-term infected cats. Two- and three-color flow cytometric analysis demonstrated that the CD8alpha+betalo subset is L-selectin negative (CD62L-) and has increased expression of CD44, CD49d, and CD18, consistent with an activation phenotype. The CD8alpha+betaloCD62L- cells but not the CD8alpha+betahiCD62L+ cells demonstrated strong antiviral activity in the FIV acute-infection assay. The progressive expansion of the CD8alpha+betaloCD62L- effector subset cells in FIV-infected cats parallels that seen in human immunodeficiency virus (HIV)-infected patients, suggesting that failure in homeostatic mechanisms regulating lymphocyte activation or trafficking (or both) may be a consequence of both HIV and FIV infections.