Regulatory effects of dexamethasone on NK and T cell immunity.

Glucocorticoids (GCs) act via the intracellular glucocorticoid receptor (GR), which can regulate the expression of target genes. With regard to the immune system, GCs may affect both innate and adaptive immunity. Our study analyzed the immunoregulatory effects of dexamethasone (Dex) treatment on splenic T, Treg, NK and NKT cells by treating C57Bl6 mice with various doses of Dex. We observed that treatment with Dex decreased the number of NK cells in the spleen and suppressed their activity. In particular, the expression of both Ly49G and NKG2D receptors was decreased by Dex. However, Dex did not affect the population of NKT cells. With regard to splenic T cells, our results show a dose-dependent reduction in CD3+, CD4+, CD8+, CD44+ and CD8+CD122+ T cells, but a stimulatory effect on CD4+CD25+ regulatory T cells by Dex treatment. In addition, treatment with Dex suppressed anti-tumor immune response in a mouse EG7 tumor model. We conclude that Dex may suppress both T- and NK-mediated immunity.

Modification of anti-tumor immunity by tolerogenic dendritic cells.

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag-/- mice, expressing a transgenic TCR in CD8+ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8+OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1+CD27- NK cells as well as Foxp3+ and IL-10 secreting CD8+OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8+ Tregs.

Local glucocorticoid production in the thymus.

Besides generating immunocompetent T lymphocytes, the thymus is an established site of de novo extra-adrenal glucocorticoid (GC) production. Among the compartments of the thymus, both stromal thymic epithelial cells (TECs) and thymocytes secrete biologically active GCs. Locally produced GCs secreted by the various thymic cellular compartments have been suggested to have different impact on thymic homeostasis. TEC-derived GCs may regulate thymocyte differentiation whereas thymocyte-derived GCs might regulate age-dependent involution. However the full biological significance of thymic-derived GCs is still not fully understood. In this review, we summarize and describe recent advances in the understanding of local GC production in the thymus and immunoregulatory steroid production by peripheral T cells and highlight the possible role of local GCs for thymus function.

The selective impact of transgenically expressed glucocorticoid receptor on T cells.

Glucocorticoids (GCs) strongly impact on different T cell subsets inducing generally immunosuppressive effects, whereas much less is known about the effect of GC on natural killer (NK) cells. The aims of this study were to investigate the effects of GC on T cell functions, including T cell-mediated anti-tumor immune response, and on NK cells. We have used lck-GR mice, which overexpress a transgenic rat GR in both T and NK cells. These mice were found to have decreased both CD4(+) and CD8(+) T cell populations in the periphery. In contrast, both NK and NKT cells were found in normal numbers in lck-GR mice. To identify genes and pathways affected by GR overexpression in our system in T cells, we have compared gene expression profiles in wild-type and lck-GR T cells. Among the genes upregulated in T cells from lck-GR mice, the microarray analysis has identified genes regulating expansion of regulatory T cells. The analysis of genes downregulated in lck-GR mice has identified genes and gene associated with the regulation of immune response. With regard to the effects on T cell functions in lck-GR mice, transgenic expression of GR had a suppressive effect on killer cell activity in vitro. In addition, lck-GR mice showed an increased tumor growth in murine tumor model in vivo, which may be a possible consequence of reduced T cell numbers and activity. We conclude that an increased expression of the GR strongly affects numbers and possibly functions of T cell subsets, but has little effect on NK cells.

Estrogen receptor α and ß in the normal immune system and in lymphoid malignancies.

Estrogens regulate various normal and pathophysiological processes including cancers. Cellular signaling by estrogens is mediated by estrogen receptor α (ERα) and ß (ERß), respectively. Binding of agonists to the ERs affects gene transcription. The main endogenous estrogen, 17ß-estradiol (E2), binds to both ERα and ERß with similar affinity. However, the ligand-binding pocket of ERα and ERß are slightly different which has allowed the development of selective ER ligands. Importantly, while estrogens via ERα stimulate proliferation, signaling via ERß inhibits proliferation and promotes apoptosis. In both normal and cancer cells the ERs are co-expressed with ER splice variants which may modify the transcriptional activity of the wild-type receptors. Estrogens have prominent effects on immune functions and both ERα and ERß are expressed in immune cells and lymphoid malignancies. With regard to lymphoid malignancies, most show estrogen influence as several epidemiological studies of lymphoid cancers demonstrate gender differences in incidence and prognosis with males being more affected. In line with these findings, recent results generated by us have shown that ERß selective agonists inhibit growth and induce apoptosis in human and murine lymphomas in vivo in xenograft experiments. This suggests that ERß selective agonists in the future may be useful in the treatment of lymphomas.

Extra-adrenal glucocorticoid synthesis: immune regulation and aspects on local organ homeostasis.

Systemic glucocorticoids (GCs) mainly originate from de novo synthesis in the adrenal cortex under the control of the hypothalamus-pituitary-adrenal (HPA)-axis. However, research during the last 1-2 decades has revealed that additional organs express the necessary enzymes and have the capacity for de novo synthesis of biologically active GCs. This includes the thymus, intestine, skin and the brain. Recent research has also revealed that locally synthesized GCs most likely act in a paracrine or autocrine manner and have significant physiological roles in local homeostasis, cell development and immune cell activation. In this review, we summarize the nature, regulation and known physiological roles of extra-adrenal GC synthesis. We specifically focus on the thymus in which GC production (by both developing thymocytes and epithelial cells) has a role in the maintenance of proper immunological function.

Keratinocyte growth factor (KGF) delays the onset of collagen-induced arthritis.

BACKGROUND: Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. KGF protects the oral and intestinal mucosa against damage induced by irradiation or chemotherapy. Previous studies have found the expression of KGF in chondrocytes and suggested that KGF promotes the wound healing process in injured cartilage. KGF also has important effects on the immune system such as the regeneration of thymus tissue and the formation of regulatory T cells (T(reg)) in the periphery. AIM: Here we investigated the effect of KGF on collagen type II induced arthritis (CIA) and anti-collagen antibody induced arthritis (CAIA) in order to discriminate between immunoregulatory effect and direct protective effect on chondrocytes. METHODS: CIA was induced by immunization with CII and CAIA by treatment of mice with a cocktail of four different anti-CII antibodies. The effect of KGF on the thymus and spleen was analyzed by FACS and by immunohistochemistry. RESULTS: We have found that KGF treatment delayed the onset of CIA but had no effect on CAIA. Our results show that KGF treatment leads both to an outflow of naïve T cells from the thymus and to a statistically significant increase in the percentage of CD4(+)Foxp3(+) T(regs) in the periphery. CONCLUSIONS: We suggest that the effect of KGF on CIA depends on immunoregulatory mechanisms. KGF may delay the aging of the cellular immune system and thus improve the resilience of the immune system against autoimmune reactions.

Thymus-derived glucocorticoids mediate androgen effects on thymocyte homeostasis.

Androgens contribute to the involution process of the aging thymus gland. However, molecular mechanisms behind this effect remain largely unknown. We have investigated the influence of testosterone on the ectopic synthesis of glucocorticoids (GCs) in thymocytes, an activity recently shown by us to be important for the homeostatic regulation of these cells. Castration, which leads to a strong increase in thymus tissue and function, was associated with a reduced GC release from thymocytes caused by down-regulated expression of several enzymes involved in GC synthesis, without affecting GC synthesis in the adrenals. Testosterone treatment of castrated male mice reversed these effects, also without affecting adrenal GC synthesis. The effects of testosterone in castrated mice on thymocyte homeostasis and GC release were strongly reduced in mice pretreated with the CYP11B1 enzyme inhibitor metyrapone, acting on the last step in the corticosterone synthesis. The androgen-induced thymic involution was dependent on GC action, because this was completely absent in mice lacking GC receptor (GR) expression specifically in thymocytes. We provide here an unrecognized mechanism how androgens contribute to thymic involution by stimulating local synthesis and release of GCs in the thymus.

Lipoprotein lipase is differentially expressed in prognostic subsets of chronic lymphocytic leukemia but displays invariably low catalytical activity.

Lipoprotein lipase (LPL) expression has been shown to correlate with IGHV mutational status and to predict outcome in chronic lymphocytic leukemia (CLL). We here investigated the prognostic impact of LPL expression in relation to other prognostic markers including IGHV3-21 usage in 140 CLL patients. Additionally, we studied the catalytic activity of LPL in CLL cells. A significant difference in LPL mRNA expression was detected in IGHV unmutated compared to mutated CLL patients (p<0.001). However, the poor-prognostic mutated/stereotyped IGHV3-21 patients did not differ from other mutated CLL cases. Clinical outcome was significantly different in CLL cases with high versus low LPL expression (p<0.001), and LPL expression exceeded mutation status/IGHV3-21 usage as an independent prognostic marker. Finally, LPL protein expression correlated significantly with mRNA expression and was higher in IGHV unmutated versus mutated CLL (p=0.018), although the majority of synthesized protein was catalytically inactive indicating a non-catalytical function in CLL.

IGHV3-21 gene usage is associated with high TCL1 expression in chronic lymphocytic leukemia.

T-cell leukemia/lymphoma protein 1 (TCL1) was recently shown to display an expression pattern in chronic lymphocytic leukemia (CLL) corresponding to molecular subtypes, where poor-risk patients demonstrated higher expression levels. Here, we examined the mRNA expression pattern of TCL1 in 144 patients with CLL, including 67 immunoglobulin heavy-chain variable (IGHV) mutated, 58 IGHV unmutated and 19 patients with IGHV3-21 usage. A higher TCL1 expression level was detected in patients with CLL with unmutated vs. mutated IGHV genes (P < 0.001), whereas no difference was demonstrated within the IGHV3-21 cohort (i.e., mutated vs. unmutated and stereotyped vs. non-stereotyped complementarity determining region 3). The IGHV3-21 subgroup displayed high TCL1 mRNA expression, differing significantly from other IGHV mutated cases (P < 0.001), although 11/19 had mutated IGHV genes. Furthermore, high TCL1 expression levels were associated with significantly shorter overall survival (P < 0.001). Altogether, we show that TCL1 mRNA expression may predict clinical outcome in CLL and that the IGHV3-21 subset, regardless of mutational status, displays high TCL1 expression.

Thymocyte-synthesized glucocorticoids play a role in thymocyte homeostasis and are down-regulated by adrenocorticotropic hormone.

Thymocytes from adult mice synthesize glucocorticoids (GCs), and some data indicate a role for this hormone production in thymic homeostasis. Here we present further support for this view by showing that the dramatic increase in thymocyte number seen after adrenalectomy (ADX) does not correlate with the decrease in systemic GCs but rather with an ACTH-mediated down-regulation of GC synthesis in thymocytes. High ACTH concentrations caused by ADX in wild-type mice down-regulated CYP11B1 mRNA expression, encoding the last enzyme required for corticosterone synthesis and as a consequence reduced GC synthesis in thymocytes. This was not seen in IL-1beta/IL-18 double-knockout mice unable to respond to ADX with high ACTH levels. However, if ADX IL-1beta/IL-18 double-knockout mice were treated with ACTH, this led to a down-regulation of CYP11B1 and GC synthesis in thymocytes. In addition, in vivo treatment of mice with the CYP11B1 antagonist metyrapone, without affecting the systemic corticosterone level, increased thymocyte numbers and in vitro treatment of isolated thymocytes prevented thymocyte loss. Furthermore, in vitro experiments showed that both ACTH and its receptor-induced second-messenger molecule cAMP down-regulated mRNA expression of critical enzymes in GC steroidogenesis and GC synthesis in thymocytes. We conclude that thymocyte-produced GCs are important for the homeostasis of adult mouse thymocytes and that high ACTH level, in contrast to stimulating GC synthesis in the adrenal glands, has the opposite effect in thymocytes.

Brefeldin A inhibits vesicular MHC class I processing in resting but not in CpG- and disruption-activated DC.

Dendritic cells (DC) can process exogenous proteins for presentation on MHC class I (MHC-I) in a vesicular pathway (VP) that is distinct from the classical, cytosolic MHC-I pathway. Here we investigate the sensitivity of this VP to Brefeldin A (BFA), a fungal metabolite which inhibits vesicular trafficking by preventing the activation of some ADP-ribosylation factor (ARF) proteins, in both resting and differently activated DC. The VP could be directly visualized in DC by the presence of OVA-derived H-2K(b)/SIINFEKL complexes in a LAMP1 positive, but EEA negative, compartment in wt but not in cathepsin S(-/-) mice as these are unable to process OVA into the SIINFEKL peptide in endolysosomes. BFA, which binds to specific ARF-GDP-sec7 sites, both in the Golgi and in endolysosomes, was found to bind to and inhibit the VP in resting DC. If the VP was selectively activated with an immunostimulatory CpG ODN, binding to endolysosomal TLR9 receptors, or by the mere mechanical disruption of clustered DC cells, BFA no longer had this effect. The activation of the VP with both CpG and cellular disruption was found to be dependent on the MyD88 adaptor protein. We conclude that vesicular MHC-I processing in DC occurs in ARF-regulated LAMP1 positive vesicles which, as a consequence of cellular activation, no longer can bind BFA and thus become resistant to the inhibitory effect of this drug.

Age-related synthesis of glucocorticoids in thymocytes.

Glucocorticoids (GCs) are primarily synthesized in the adrenal glands but an ectopic production has also been reported in the brain, the gastrointestinal tract and in thymic epithelial cells (TEC). Here we show that thymocytes express genes encoding for all enzymes required for de novo GC synthesis and produce the hormone as demonstrated by both a GC specific reporter assay and a corticosterone specific ELISA assay. Interestingly, GC synthesis is detectable in cells from young mice (4 weeks) and thereafter increases during aging (14-22 weeks) together with an increased gene expression of the rate-limiting enzymes StAR and CYP11A1. Hormone production occurred at a thymocyte differentiation stage characterized by being double positive for the CD4 and CD8 surface markers but was found to be unrelated to CD69 expression, a marker for thymocytes undergoing positive selection. No GC synthesis was found in resting or anti-CD3 activated CD4 and CD8 positive T cells isolated from the spleen. Thymocyte-derived GC had an anti-proliferative effect on a GR-transfected cell line and induced apoptosis in thymocytes. The age- and differentiation stage-related GC synthesis in thymocytes may play a role in the involution process that the thymus gland undergoes.

Adenosine, through the A1 receptor, inhibits vesicular MHC class I cross-presentation by resting DC.

Levels of the purine nucleoside adenosine (Ado) increase during conditions related to hypoxia such as inflammation, tissue damage and cancer. Ado binds to a set of G protein-linked receptors (A(1), A(2A), A(2B) and A(3)) which are widely and differentially expressed in tissues and regulate inflammatory and autoimmune responses. We have investigated the effect of the stable Ado analogue 2-Chloro-Adenosine (2-Cl-Ado) on vesicular MHC class I cross-presentation using the exogenous protein ovalbumin (OVA) and cultured mouse dendritic cells (DC) at different stages of maturation and activation. 2-Cl-Ado was found to strongly inhibit cross-presentation of OVA by resting DC (rDC) but had a much smaller effect on immature and CpG-activated DC. The effect of Ado on rDC could be fully reversed by the Ado receptor antagonist CGS 15943 and by pertussis toxin demonstrating that it was mediated by a Gi-linked Ado receptor. A(1) Ado receptor mRNA was found to be upregulated in rDC and, by using rDC from A(1), A(2A) and A(3) receptor knockout mice, this receptor was found to mediate most of the suppression. 2-Cl-Ado did not influence the cellular uptake of OVA, the cytosolic processing of the protein in rDC or the formation of intracellular MHC-I/peptide complexes in a LAMP1 positive vesicular compartment but inhibited the transport of these to the cell surface. It is concluded that 2-Cl-Ado, by acting on A(1) receptor, can suppress vesicular MHC class I cross-presentation in rDC.

Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus, and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor.

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8(+) T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8(+) T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8(+) T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8(+) T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L(+) P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8(+) T cell response correlates with tumor protection.

Interference with O-glycosylation in RMA lymphoma cells leads to a reduced in vivo growth of the tumor.

Carbohydrate processing in cancer cells can influence the growth, metastatic potential, vascularization and immune recognition of such cells. Interference with N-glycosylation has been shown both to reduce the membrane expression of MHC class I and to increase the in vitro sensitivity of tumor cells to NK cell killing. We investigated the effect of O-glycosylation inhibition on the in vivo growth, phenotype and NK sensitivity of RMA lymphoma cells using benzyl N-acetyl-alpha-D-galactosamide (BAG). BAG-treated cells were found to have a strongly reduced local growth potential in vivo. However, inhibition of O-glycosylation caused this effect without any significant downregulation of MHC-I and increase in sensitivity to NK killing as seen after inhibition of N-glycosylation using Castanospermine. BAG treatment of RMA cells resulted in the removal of larger O-linked glycans and a high expression of the T-antigen (GalGalNAc), a target for natural antibodies (NAs) induced by the gastrointestinal bacterial flora. Whether the loss of larger O-linked glycans, and associated functions, or of biological effects of NA contributed to the antitumor effect remains to be established. The results support the idea that inhibitors of O- as well as N-linked glycosylation may be useful for the treatment of cancer, given that they can be specifically targeted to the tumor tissue.

Conditional expression of a glucocorticoid receptor transgene in thymocytes reveals a role for thymic-derived glucocorticoids in thymopoiesis in vivo.

We and others have previously reported that thymic epithelial cells produce glucocorticoids (GCs). In vitro studies have also suggested that thymic-derived GCs play a role in the development of thymocytes. However, until now it has not yet been established whether thymic-derived GCs play a role in thymopoiesis in vivo. To investigate this, we conditionally overexpressed the GC receptor (GR) in thymocytes using transgenic mice with a tetracycline-inducible expression system. The influence of systemic GCs was excluded by adrenalectomizing the transgenic mice before the GR induction. Conditional expression of transgenic GR in the thymocytes of adrenalectomized transgenic mice led to a decrease in the thymocyte number. This was associated with increased thymocyte apoptosis. The effect of thymic-derived GCs on the thymocytes was confirmed after transgenic GR induction in a thymic organ culture system. Finally, the GR antagonist RU486 increased thymocyte number in adrenalectomized mice in vivo and prevented a reduction in thymocyte number in thymic organ culture after transgenic GR induction. These observations further confirmed a role for the thymic-derived GCs in regulating thymocyte homeostasis in vivo.

High expression of bfl-1 contributes to the apoptosis resistant phenotype in B-cell chronic lymphocytic leukemia.

In order to identify regulatory genes involved in the development of an apoptosis-resistant phenotype in patients with chemotherapy refractory B-cell chronic lymphocytic leukemia (B-CLL) expression of apoptosis-regulating genes in B-CLL cells was quantified using cDNA arrays and RT-PCR. Data were obtained from and compared between 2 groups of B-CLL patients with either nonprogressive, indolent, previously untreated disease and with leukemic cells sensitive to in vitro fludarabine-induced apoptosis, referred to as sensitive B-CLL (sB-CLL) or with progressive, chemotherapy refractory disease and with leukemic cells resistant to in vitro fludarabine-induced apoptosis, referred to as resistant B-CLL (rB-CLL). By performing a supervised clustering of genes that most strongly discriminated between rB-CLL vs. sB-CLL a small group of genes was identified, where bfl-1 was the strongest discriminating gene (p < 0.05), with higher expression in rB-CLL. A group of apoptosis-regulating genes were modulated during induction of apoptosis by serum deprivation in vitro in a similar manner in all cases studied. However, bfl-1 was preferentially downregulated in sB-CLL as compared to rB-CLL (p < 0.05). We conclude that bfl-1 may be an important regulator of B-CLL apoptosis, which could contribute to disease progression and resistance to chemotherapy, and as such represent a future potential therapeutic target.

Different roles for glucocorticoids in thymocyte homeostasis?

Glucocorticoids (GCs) have important immunoregulatory effects on thymocytes and T cells. Ectopic production of GCs has been demonstrated in thymic epithelial cells (TECs) but the role of GCs in thymocyte homeostasis is controversial. Studies in several different mouse models, genetically modified for the GC receptor (GR) expression or function, have demonstrated conflicting results in terms of the effect of the hormone on thymocytes. Here, we summarize these data and suggest that GCs can mediate both positive and negative effects in the organ depending on the local hormonal concentration. Basal GC levels might promote growth of early thymocytes in young mice, and increased levels, generated through a stress reaction, apoptosis in these cells. A gradual loss of GC synthesis in TECs during aging might contribute to thymic involution, a process so far unexplained.

Loading of the antigen-presenting protein CD1d with synthetic glycolipids.

CD1 proteins present mammalian and microbial lipid and glycolipid antigens to different subsets of T cells. Few such antigens have been identified and the binding of these to CD1 molecules has mainly been studied by using responding T cells in cellular assays or recombinant solid-phase CD1 proteins. In the present study we use four different glycolipids, some of which contain tumor-associated carbohydrate antigens, to develop a procedure to easily detect binding of glycolipids to CD1 proteins on viable cells. Two of these glycolipids are novel glycoconjugates containing alpha-D-N-acetylgalactosamine (alpha-GalNAc) that were prepared by a combined solution and solid-phase approach. The key step, a Fischer glycosylation of 9-fluorenylmethoxycarbonylaminoethanol with GalNAc, furnished the alpha-glycoside 4 in 34% yield. Cells were incubated with glycolipids and stained with monoclonal antibodies specific for the carbohydrate part. The level of glycolipid bound to cells was then determined by flow cytometry with a secondary antibody labeled with fluorescein isothiocyanate. All four glycolipids were found to bind to CD1d but with different selectivity. The loading was dose dependent and could be inhibited by an established CD1d ligand, alpha-galactosylceramide. Through use of this procedure, glycolipids were selectively loaded onto CD1d expressed on professional antigen-presenting cells for future use as cellular vaccines. Moreover, the glycolipids described in this study represent novel CD1d-binding ligands that will be useful derivatives in the study of CD1d-dependent immune responses, for example, against tumors.