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Interleukin-4 (IL-4) is a cytokine secreted by interleukin-2 (IL-2)-activated lymphocytes. IL-2stimulated lymphocytes also secrete two cytokines, tumour necrosis factor (TNF) and gammainterferon (IFN-γ), which contribute to effector function and which may themselves recruit fresh, cytokine-secreting effector cells. We have now investigated whether the IL-4 induced is able to homeostatically regulate secretion of the TNF and IFN-γ. Peripheral blood mononuclear cells or lymphocytes from normal donors and from patients with neoplastic disease were cultured in the presence of IL-2 alone, IL-4 alone or with both cytokines. IL-2 induced high levels of TNF and IFN-γ secretion in both groups. The addition of recombinant IL-4 to these IL-2-stimulated cultures lead to significant inhibition of IFN-γ and TNF production. IFN-γ, secretion was reduced by 50-99% in normal donors and by between 11% and 99% in patients (P < 0.001). TNF levels induced by IL-2 were similarly reduced by IL-4 both in normal donors (P<0.003) and in patients (P<0.01). These inhibitory effects were produced by IL-4 at doses of IL-2 attainable in vivo. Inhibition appears to represent a homeostatic regulatory mechanism which may limit recruitment of fresh activated killer (AK) cells. When endogenous IL-4 activity in IL-2-activated lymphocytes was blocked by anti-IL-4 antibody, significantly higher levels of IFN-γ, and TNF were secreted (P < 0.05). Since both TNF and IFN-γ may contribute to the anti-neoplastic action of IL-2, manipulating the level of IL-4 activity in vivo could augment the benefits of IL-2 immunotherapy. [ABSTRACT FROM AUTHOR]

CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA(+) T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.

p40, the common subunit of the proinflammatory cytokines IL-12 and IL-23, is produced by resident skin cells. Whereas the in vivo effects of IL-12 are well established, little is known about the role of IL-23 in cutaneous immune responses. In this study we show that p40 transgenic (TG) mice constitutively produce IL-23 (p19/p40), but not IL-12 (p35/p40), in basal keratinocytes by cosecretion of TG p40 with endogenous p19. Repeated injections of rIL-23 in littermate (LM) mice result in an inflammatory skin disease similar to that of p40 TG mice, confirming the proinflammatory activity of IL-23. Furthermore, IL-23 secretion by p40 TG keratinocytes induces elevated numbers of Langerhans cells (LC) with a marked up-regulation of costimulatory molecules, indicating advanced maturation of keratin 14 (K14)/p40 LC when compared with LM LC. At the functional level, freshly isolated K14/p40 LC greatly exceeded LC from LM animals in their capacity to stimulate allogeneic T cell proliferation. To assess whether IL-23 regulates cutaneous immune responses in vivo, we used an allogeneic skin transplantation model. Full thickness skin grafts from K14/p40 donors (H-2(q)) transplanted across a MHC class I and class II barrier onto BALB/c (H-2(d)) recipients were rejected in a significantly accelerated fashion (mean survival time: 8.8 days) when compared with skin grafts from non-TG LM (H-2(q)) (mean survival time: 10.7 days, p < 0.01). Based on these results we propose that IL-23-induced changes of LC may be an important mechanism in directing the outcome of cutaneous immune responses.

Expression of CD68 (macrosialin) in the absence of surface and lysosomal lineage marker molecules is a characteristic feature of T zone-associated plasmacytoid monocytes, which were recently shown to represent precursors of dendritic cells (DC). We demonstrate here a minor population of strongly CD68-positive (CD68bright) blood cells that lack all analyzed myeloid surface (CD14-, CD33-, CD13-, CD11b-, CD11c-) and lysosomal (myeloperoxidase, MPO- and lysozyme, LZ-) marker molecules (0.4 +/- 2% of the total mononuclear cells). These CD68bright, lineage marker-negative (lin-) cells can be induced to proliferate in the presence of IL-3. They do not acquire myeloid features even upon stimulation with granulocyte-macrophage CSF plus IL-1, IL-3, and IL-6. Instead, these cells develop typical DC characteristics upon culture. Furthermore, these CD68brightlin- DC precursors acquire mature DC characteristics (CD86+, CD83+, CD54bright) upon stimulation with CD40 ligand plus IL-3. A second subset of DC precursor-like blood cells was found to weakly express CD68 (0.3 +/- 0.2% of the total mononuclear cells) and to coexpress several myeloid lineage associated molecules (LZ+, CD11c+, CD33+, CD13+). Cells of this second subset resemble both previously described myeloid-related peripheral blood DC and germinal center DC. Analysis of peripheral blood leukocytes for CD68 thus revealed the existence of two cell subsets that phenotypically resemble lymphoid tissue-associated DC. The unique phenotype CD68brightlin- is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68brightlin- blood leukocytes also functionally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68brightlin- blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway.

Epidermal Langerhans cells (LC) play a critical role in host defense. Still we know rather little about the development and functional specialization of these bone marrow-derived dendritic cells (DC) located in the most peripheral ectodermal tissue of the mammalian organism. How LC develop from their primitive progenitors in bone marrow and to what extent LC are related in their development to other lineages of the hemopoietic system is still under debate. There are currently 3 major areas of debate: 1) which are the signals required for LC development and differentiation to occur, 2) what are the (molecular) characteristics of the intermediate stages of LC differentiation, and 3) how are LC related in their development and/or function to other cells of the hemopoietic system? A better understanding of LC development and answers to these questions can be expected from recently developed technologies which allow the in vitro generation of DC with the typical molecular, morphological and functional features of LC from purified CD34+ progenitor cells under defined serum-free culture conditions. TGF-beta 1 was found to be an absolute requirement for in vitro LC development under serum-free conditions upon stimulation with the classical DC growth and differentiation factors GM-CSF, TNF-alpha and SCF. The recently identified cytokine FLT3 ligand further dramatically enhanced in vitro LC development and even allowed efficient in vitro generation of LC colonies from serum-free single cell cultures of CD34+ hemopoietic progenitor cells.

A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34+ cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34+ CB cells were stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7+/-11.5 fold vs. 22.5+/-4.7 fold without FL) and generation of CD1a+ DCs (mean 45.7+/-9.8% vs. 28+/-6.5% without FL, n = 4,p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+LNGFR+ cells (mean 10%+/-4.4% vs. 6%+/-2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a+ DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a+CD80+ and CD1a+CD86+ DCs after 12-14 days of culture. In addition, transduced CD1a+ DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a+ DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a+ DCs derived from CD34+ progenitor cells and may be very useful for future therapeutic applications of DCs.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder described as a clinical triad of thrombocytopenia, eczema, and immunodeficiency. The gene responsible for WAS encodes a 502-amino acid proline-rich protein (WASp) that is likely to play a role in the cytoskeleton reorganization and/or in signal transduction of hematopoietic cells. However, the function and the regulation of the WAS gene (WASP) have not yet been clearly defined. We have studied WASP expression at the transcriptional level in freshly isolated mature peripheral blood cells and during hematopoietic development. For this purpose, we have isolated CD34+ hematopoietic precursor cells from cord blood. These cells were cultured in vitro with various growth factors to generate committed or mature cells belonging to different hematopoietic differentiation pathways, such as granulocytic (CD15+) cells, monocytic (CD14+) cells, dendritic (CD1a+) cells, erythroid lineage (glycophorin A+) cells, and megakaryocytic cells (CD41+). We have shown by reverse transcriptase polymerase chain reaction analysis that the WASP transcript is ubiquitously detectable throughout differentiation from early hematopoietic progenitors, including CD34+CD45RA- and CD34+CD45RA+ cells, to cells belonging to different hematopoietic lineages, including erythroid-committed and dendritic cells. In addition, Northern blot analysis showed that peripheral blood circulating lymphocytes (CD3+ and CD19+ cells) and monocytes express WASP mRNA. Several hematopoietic cell lines were tested and higher levels of expression were consistently detected in myelomonocytic cell types. By contrast, primary nonhematopoietic cells, including fibroblasts, endothelial cells, and keratinocytes, were consistently negative for WASP mRNA.

Leukemic relapse is the major complication following autologous bone marrow transplantation (BMT) in acute myeloblastic leukemia (AML). Previously, we have shown that recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) infusion after autologous BMT has the ability to augment endogenous activated killer (AK) cell function which may play a role in the eradication of minimal residual disease. However, the clinical application of rhGM-CSF in patients with AML has been limited by its potential stimulatory effect on the malignant clone. Here we report the effect of rhGM-CSF 5 micrograms/kg/day infusion on AK cell function in 20 patients with AML undergoing autologous BMT. AK cell function was investigated before autologous BMT, during rhGM-CSF therapy and after withdrawal. In addition, its influence on the actuarial risk of relapse is analyzed and compared with a historical control group of 20 patients transplanted immediately before initiation of this study. rhGM-CSF significantly enhanced AK cell function. During rhGM-CSF treatment, median AK cell function rose from 1.8% before autologous BMT (range 0-8%) to 35% (range 3-80%) and remained increased after cessation of rhGM-CSF (median 20%; range 0-36%; P < 0.001). After a median follow-up of 24 months, the actuarial risk of relapse is 37.4% in rhGM-CSF-treated patients compared with 49.5% in controls (P = 0.05). Interestingly, none of the 7 patients with an AK cell activity > or = 20% in the first 2-5 weeks after autologous BMT have relapsed compared with 6 of 9 patients with an AK cell activity < 20% (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

The c-myc oncogene c-Myc is commonly activated in cancer and transactivates gene expression by binding to CACGTG DNA sequences as a heterodimeric complex with Max. The ornithine decarboxylase (ODC), p53, prothymosin alpha and ECA39 promoters are transactivated by c-Myc, and are considered direct targets, as activation is mediated by CACGTG sequences. Interestingly, the c-Myc-responsive CACGTG sequences in the p53, prothymosin alpha, ECA39 and murine ODC genes are all downstream of the RNA CAP site, suggesting that downstream sequences are preferred c-Myc targets. Using a series of heterologous reporter constructs, we have tested the effects of position and orientation of c-Myc-responsive CACGTG sequences on c-Myc's ability to activate transcription. A single binding site conferred c-Myc-responsiveness independent of position and orientation, and over distances of 1.7 kbp. The extent of transactivation was not significantly influenced by position of the responsive elements. By contrast, the extent of transactivation was dependent upon the number of c-Myc binding sites. The results demonstrate that c-Myc activates transcription independent of position and orientation and that considerable flexibility exists in the interaction of c-Myc transactivation domains with the general transcription machinery.

The interactions of GM-CSF with cells of lymphoid lineage are not well understood and their clinical use has been focused on the acceleration of hematopoietic recovery. However, several reports have shown that human GM-CSF can affect certain T lymphocyte in vitro cytotoxic functions. To assess whether recombinant human GM-CSF (rhGM-CSF) has a more broadly based activity in the immune system, we studied its in vivo effects on endogenously-generated killer function in patients undergoing ABMT for hematologic malignancies. Eleven patients received rhGM-CSF after ABMT: eight received rhGM-CSF as a 2-h infusion daily from days +3 to +17 and three received rhGM-CSF until reaching > 500 x 10(6)/l granulocytes. Eight patients not enrolled in the rhGM-CSF therapy protocol served as controls. Natural killer (NK) cell activity and activated killer (AK) cell activity were studied before conditioning, during rhGM-CSF therapy and after withdrawal of GM-CSF. rhGM-CSF therapy does not affect NK activity. Circulating lymphocytes with the ability to kill AK-sensitive targets appear spontaneously in control ABMT patients. AK activity was 1.6 +/- 0.8% before ABMT increasing to 9 +/- 2.5% and 14 +/- 2.1% at 2 and 3 weeks after ABMT, respectively (p = 0.002). In rhGM-CSF-treated patients this phenomenon also occurs. AK activity increased from 2.4 +/- 1.5% before ABMT to 33.6 +/- 8.1% during rhGM-CSF administration (p = 0.001) and 17.5 +/- 3.4% after withdrawal (p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-4 (IL-4) is a cytokine secreted by interleukin-2 (IL-2)-activated lymphocytes. IL-2-stimulated lymphocytes also secrete two cytokines, tumour necrosis factor (TNF) and gamma-interferon (IFN-gamma), which contribute to effector function and which may themselves recruit fresh, cytokine-secreting effector cells. We have now investigated whether the IL-4 induced is able to homeostatically regulate secretion of the TNF and IFN-gamma. Peripheral blood mononuclear cells or lymphocytes from normal donors and from patients with neoplastic disease were cultured in the presence of IL-2 alone, IL-4 alone or with both cytokines. IL-2 induced high levels of TNF and IFN-gamma secretion in both groups. The addition of recombinant IL-4 to these IL-2-stimulated cultures lead to significant inhibition of IFN-gamma and TNF production. IFN-gamma secretion was reduced by 50-99% in normal donors and by between 11% and 99% in patients (P less than 0.001). TNF levels induced by IL-2 were similarly reduced by IL-4 both in normal donors (P less than 0.003) and in patients (P less than 0.01). These inhibitory effects were produced by IL-4 at doses of IL-2 attainable in vivo. Inhibition appears to represent a homeostatic regulatory mechanism which may limit recruitment of fresh activated killer (AK) cells. When endogenous IL-4 activity in IL-2-activated lymphocytes was blocked by anti-IL-4 antibody, significantly higher levels of IFN-gamma and TNF were secreted (P less than 0.05). Since both TNF and IFN-gamma may contribute to the anti-neoplastic action of IL-2, manipulating the level of IL-4 activity in vivo could augment the benefits of IL-2 immunotherapy.

Administration of interleukin 2 (IL2) to patients with minimal residual malignant disease following myeloablative chemo-radiotherapy may augment immune reconstitution and reduce the risk of relapse by increasing cytotoxic effector function and cytokine dependent killing directed at residual malignant cells. The ability of IL2 generated activated killer cells to inhibit haemopoietic progenitor cells and to release gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) may, however, retard haemopoietic recovery, as both TNF and gamma IFN inhibit normal myelopoiesis in vitro. To determine the effect of IL2 infusion on myeloid regeneration in vivo, we have examined haemopoietic recovery in patients receiving this cytokine following autologous marrow transplantation or ablative chemotherapy. We find that IL2 infusion accelerates neutrophil recovery and that granulocyte-macrophage colony stimulating factor (GMCSF) and IL3 mRNA become detectable in circulating mononuclear cells. Induction of TNF by IL2 may also contribute to subsequent acceleration of myelopoiesis by initiation of GM-CSF mRNA synthesis in patient marrow fibroblasts. These results show that IL2 infusion may facilitate myeloid recovery when administered during the period of haemopoietic regeneration following ablative chemoradiotherapy.

Culture of lymphocytes from allograft recipients or from normal donors with Interleukin 2 (IL2) induces high levels of gamma-interferon (gamma IFN) and tumour necrosis factor (TNF) secretion. We now show that production of these two cytokines can also be increased after autologous bone marrow transplantation (BMT) and induced following chemotherapy for haematological malignancy. These effects occur even though the regenerating IL2 responsive lymphocytes in this context have previously been extensively exposed to cytotoxic agents. IL2 induced secretion of gamma-IFN and TNF is higher in autograft recipients than in patients treated by chemotherapy alone. This effect may be related to differences in the phenotypic profile between recovering lymphocytes in the two groups and to an increased degree of prior in vivo lymphocyte activation in the autologous bone marrow transplant recipients. In both groups of patients, IL2 acts on CD3+ T cells and on CD16+ NK cells so that depletion of either subset incompletely abrogates IL2 dependent gamma-IFN secretion. As both TNF and gamma-IFN possess anti-leukaemic and anti-infective activity, enhancement of their secretion by IL2 infusion after autologous bone marrow transplant and induction after chemotherapy may be of therapeutic benefit.