IL-3-producing basophils are required to exacerbate airway hyperresponsiveness in a murine inflammatory model.

BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.

Activation of basophils by the double-stranded RNA poly(A:U) exacerbates allergic inflammation.

BACKGROUND: It is commonly acknowledged that asthma is exacerbated by viral infections. On the other hand, basophil infiltration of lung tissues has been evidenced postmortem in cases of fatal disease, raising the question of a possible link between these two observations. OBJECTIVES: Herein, we addressed the relationship between asthma exacerbation by viral infection and basophil activation and expansion by investigating how stimulation with the dsRNA polyadenylic/polyuridylic acid [poly(A:U)] affected basophil activities and recruitment in an allergic airway inflammation model. METHODS: The effect of dsRNA on basophils was assessed by measuring the cytokine levels produced upon stimulation. We used an OVA-induced experimental model of allergic asthma. Airway hyperreactivity, recruitment of infiltrating cells, and cytokine production were determined in the lung of mice having received poly(A:U), as compared with untreated controls. The exacerbating effect of basophils was assessed both by adoptive transfer of poly(A:U)-treated basophils and by their in vivo depletion with Ba103 antibody. RESULTS: We found that in vitro treatment with poly(A:U) increased basophil functions by inducing TH 2-type cytokine and histamine production, whereas in vivo treatment increased peripheral basophil recruitment. Furthermore, we provide the first demonstration for increased infiltration of basophils in the lung of mice suffering from airway inflammation. In this model, disease symptoms were clearly exacerbated upon adoptive transfer of basophils exposed to poly(A:U), relative to their unstimulated counterpart. Conversely, in vivo basophil depletion alleviated disease syndromes, thus validating the transfer data. CONCLUSIONS: Our findings provide the first evidence for airway inflammation exacerbation by basophils following dsRNA stimulation.

Expression and localization of alpha-fetoprotein mRNA and protein in human early villous trophoblasts.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Fas cross-linking mimics the inhibitory effect of anti-CD3 on IL-3-induced histamine and cytokine production by murine myeloid spleen cell precursors.

In the present study we investigated the effect of anti-CD3 stimulation on IL-3-induced histamine, IL-6, and IL-4 synthesis by murine hematopoietic precursor cells. These activities were strikingly decreased in splenocytes from mice that had received a single intravenous injection of 10 microg of anti-CD3 monoclonal antibody (mAb) 24 hours previously. A similar inhibition occurred after 24-hour in vitro stimulation of normal spleen cells with 1 microg/mL of anti-CD3 mAb. In both situations the inhibitory effect depended on T cell activation in that treatment with F(ab')2 fragments of anti-CD3 did not diminish secretion of histamine and cytokines. Cross-linking of Fas antigen on spleen cells mimicked the action of anti-CD3, provided that interferon (IFN)-gamma was present during the incubation period. Substantial amounts of this cytokine were detected in spleen cell supernatants, which were able to replace recombinant IFN-gamma during Fas receptor cross-linking. This effect was entirely mediated by IFN-gamma, as assessed by its neutralization in the presence of anti-IFN-gamma mAbs. In contrast to splenocytes, bone marrow cells responded normally to IL-3 after in vivo or in vitro stimulation with anti-CD3. They were also not affected by combined treatment with anti-Fas mAb and IFN-gamma. Together, our data support the notion that the decrease in IL-3-induced histamine and IL-6 production by splenocytes pretreated with anti-CD3 is mediated, at least in part, by Fas/FasL interactions, suggesting that the activity of extramedullary myeloid precursor cells can be modulated by molecules involved in apoptosis.

Murine hematopoietic progenitor cells produce IL-6 in response to IgE.

Similarly to interleukin-3 (IL-3), IgE is capable of inducing IL-6 production by murine bone marrow cells (BMC). IgE responder cells do not belong to the mature bone marrow compartment but coenrich with hematopoietic progenitors in the low-density fraction of a discontinuous Ficoll gradient. A significant enhancement of IL-6 production is observed after a 4-hour stimulation, reaching a maximum between 24 and 48 hours and is preceded by increased mRNA expression. The effect of IgE on IL-6 production is not mediated by IL-3 since it is not modified by anti-IL-3 antibodies. Upon a 4-hour exposure to IgE or IL-3, a similar percentage of progenitor-enriched BMC expresses IL-6 mRNA (3.9 and 5.4%, respectively, as determined by in situ hybridization), which is not further increased by a combination of both stimuli. IgE and IL-3 responder cells also cannot be distinguished on the basis of size, internal structure, and rhodamine (Rh) retention. The BMC sorted in the most fluorescent Rhbright subset (approximately 0.2% of total BMC) produce 30- to 40-fold more IL-6 than unfractionated cells and are similarly enriched for CFU-cells (CFU-C). The most primitive cells concentrated in the Rhdull fraction do not express this biological activity. The sorted Rhbright population does not contain mature mast cells/basophils or monocytes, and IL-6 is not produced in response to Fc epsilon RI cross-linkage after presensitization with IgE.

Modulation of histidine decarboxylase activity and cytokine synthesis in human leukemic cell lines: relationship with basophilic and/or megakaryocytic differentiation.

In the present study, we show that UT7D1 cells, derived from the pluripotent cell line UT7, express high levels of histidine decarboxylase (HDC) mRNA spontaneously. These cells conserve the ability to differentiate into megakaryocytes upon stimulation with PMA, while greatly increasing their HDC activity. We provide evidence that enhanced HDC activity reflects the basophil rather than the megakaryocytic differentiation potential of UT7DI cells. Indeed, in addition to HDC mRNA, they express spontaneously several other mRNA coding for molecules present in basophils (FcepsilonRI, CCR3, IL-4Ralpha, IL-5Ralpha). Furthermore, the basophil antigen Bsp-1 is displayed on the surface of some UT7D1 cells in response to PMA concomitantly with increased histamine synthesis and mRNA expression of typical basophil-derived cytokines (IL-6, IL-4, and IL-13). Nevertheless, PMA cannot sustain the differentiation of this lineage, because mRNAs for basophil markers gradually diminish during long-term culture, whereas molecules associated with the megakaryocytic lineage remain prominent. In support of the notion that HDC activity is not related with megakaryopoiesis, we show that PMA-induced CD41 expression and PDGF transcription occurs in the K562 cells, though neither HDC mRNA nor any known basophil marker are expressed in these conditions. In contrast, all these markers are expressed in the basophilic leukemia cell line KU812F. Interestingly, the megakaryocytic cell line HEL produces also substantial amounts of histamine and expresses FcepsilonRI, thus revealing its basophil differentiation potential. HEL as well as KU812F need not be stimulated with PMA to react with Bsp-1 mAb, suggesting that they are more engaged into the basophil differentiation scheme than UT7D1. Other leukemic cell lines unrelated to the megakaryocyte or basophil lineage, like HL60 and U937 do neither synthesize histamine nor express basophil markers before or after PMA stimulation. To our knowledge, this is the first evidence for a factor-dependent cell line with megakaryocyte/basophil bipotentiality with which early stages of basophil commitment can be analyzed.

IL-3-induced coexpression of histidine decarboxylase, IL-4 and IL-6 mRNA by murine basophil precursors.

Murine low-density bone marrow cells sorted from the blast cell window on the basis of high rhodamine-123 retention (Rh-bright), are highly enriched in histamine-, IL-4-, and IL-6-producing cells. We established by in situ hybridization that up to 50% of this population (around 0.25% of the whole bone marrow) coexpressed the transcripts for these molecules upon stimulation with 1L-3. Rh-bright cells were also positive for mRNA encoding the alpha, beta, and gamma chains of the Fc(epsilon)RI which was functional since aggregated IgE induced the same percentage of cells hybridizing with the HDC probe as IL-3. Clonogenic progenitors and histamine- and cytokine-producing cells copurified in the Rh-bright population, but could be distinguished by their c-kit expression, CFU-C being more frequent in the c-kit(high) fraction, while histamine and IL-6 producers were enriched in the kit(low) counterpart. Ultrastructural analysis of Rh-bright cells revealed essentially two subsets, namely undifferentiated blast cells and basophil precursors. No other lineage-committed population was enriched by this sorting procedure, and it can therefore be concluded that coexpression of HDC, IL-6, and IL-4 transcripts in response to IL-3 or aggregated IgE takes place mainly in hematopoietic precursors belonging to the basophil lineage.

Role of interleukin-2 receptors in the suppressive effect of spleen cells from anti-CD3-treated mice.

In the present study, we demonstrate that unresponsive spleen T cells from mice injected with a low dose of anti-CD3 mAb (single 10 micrograms i.v. injection) significantly inhibit Con A-induced proliferation of normal spleen cells. The induction of this phenomenon requires in vivo activation since spleen cells from mice injected with the F(ab')2 fragment of anti-CD3 mAb fail to promote it. Suppression of normal T cell proliferation is concomitant with increased expression of IL-2 receptor on spleen cells from anti-CD3-treated mice. It disappears within 3 days when IL-2R has returned to background levels. A normal proliferative response to Con A can be restored when high concentrations of IL-2 are added together with the "suppressor" cells. Taken together, these data support the notion that activated spleen cells from anti-CD3-injected mice exert their inhibitory effect by competing for the IL-2 generated during culture.

[Effects of hematopoietic growth factor (GM-CSF: granulocyte-macrophage colony-stimulating factor) on thymocyte proliferation induced by interleukin-1 (IL-1)]. / Effet d'un facteur de croissance hématopoiétique (GM-CSF: granulocyte-macrophage colony-stimulating factor) sur la prolifération thymocytaire induite par l'interleukine-1 (IL-1).

A semi-purified fraction obtained from P388 D1 cell line conditioned medium (P388 D1 CM) which contains Interleukin-1 (IL-1) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) stimulates murine thymocyte proliferation both in the absence and the presence of a suboptimal dose of phytohemagglutinin (PHA). Because this effect on thymocyte proliferation is always larger than that obtained with optimal concentrations of pure IL-1, we have investigated the possible involvement of GM-CSF in this semi-purified fraction mediated-thymocyte proliferation. We here show that the maximal level of thymocyte proliferation induced by the semi-purified fraction is comparable to that obtained by the co-addition of recombinant GM-CSF and IL-1. In addition, although GM-CSF alone induces no significant thymocyte proliferation, the presence of an anti-GM-CSF antiserum partially blocks the thymocyte proliferation induced by the semi-purified fraction. Thus, the capacity of the semi-purified fraction of P388 D1 to stimulate thymocyte proliferation appears to result from a synergistic action between GM-CSF and IL-1.

Potentiating effect of granulocyte-macrophage colony-stimulating factor on interleukin-1-induced thymocyte proliferation: evidence for an interleukin-2 and tumor necrosis factor-independent pathway.

The effect of Colony-Stimulating Factors (CSFs) on the growth of murine thymocytes was investigated. None among the following factors tested alone, i.e., Interleukin-3 (IL-3), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Granulocyte Colony-Stimulating Factor (G-CSF) or Macrophage Colony-Stimulating Factor (M-CSF) has been found to stimulate thymidine uptake by thymocytes. However, GM-CSF synergistically enhances thymocyte proliferation induced by Interleukin-1 (IL-1). Synergistic responses are obtained at a very pronounced level after 3 days of culture with very low factor concentrations (1.5 to 15 pM) and in the complete absence of mitogen. Similar effects are induced by IL-3, though to a lesser degree. In contrast, neither G-CSF nor M-CSF potentiate thymocyte proliferation promoted by IL-1. Kinetic studies show that the synergy between IL-1 and GM-CSF reaches its maximum after about 72 h of thymocyte culture and that it requires the simultaneous presence of both factors during the first 24 h. In addition, our data suggest that GM-CSF acts in synergy with IL-1 by an Interleukin-2 (IL-2)-independent pathway since: (i) incubation of thymocytes with GM-CSF in the presence of IL-1 does not significantly enhance the expression of the IL-2 receptors (IL-2R) as demonstrated by flow cytometry, and, (ii) specific monoclonal antibodies against murine IL-2 or IL-2R fail to reduce thymocyte proliferation in response to the synergistic combination. Similarly, the potentiating effect of GM-CSF on IL-1 thymocyte growth does not depend on Tumor Necrosis Factor alpha (TNF) since (i) the synergy for IL-1 and GM-CSF and that previously described for IL-1 and TNF cumulate and (ii) anti-TNF antibodies do not abolish the potentiating effect of GM-CSF.

Interleukin 3 promotes histamine synthesis in hematopoietic progenitors by increasing histidine decarboxylase mRNA expression.

Interleukin 3 (IL-3) is a potent stimulator of histamine production by cells from murine hematopoietic organs. We demonstrate herein that this phenomenon results from increased histidine decarboxylase (HDC: EC 4.1.1.22) activity in progenitor-enriched bone marrow cells (around 5% of the total bone marrow) isolated from the low density layers of a discontinuous Ficoll gradient. HDC levels are markedly enhanced after a 24 h incubation with IL-3 while a 4 h exposure results only in a slight activation. It results from increased expression of the mRNA coding for HDC, as assessed by Northern blot analysis after a 24 h incubation with IL-3. At the same time point and after a 4 h stimulation, we have evaluated the percentage of cells in this population which express HDC mRNA in response to IL-3, using in situ hybridization with the antisense riboprobe. We have thus established that enhanced HDC mRNA expression occurs in a small immature subset representing from 5 to 8% of the progenitor-enriched bone marrow cells.

IL-7 is requisite for IL-1-induced thymocyte proliferation. Involvement of IL-7 in the synergistic effects of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor with IL-1.

In the absence of artificial comitogens murine thymocytes proliferate significantly in response to IL-1 at high but not at low cell densities. This observation has led us to examine a possible indirect mechanism requiring other thymocyte-growth factors, such as IL-2, IL-4, IL-6, and IL-7, in this phenomenon. Our data provide evidence that IL-7 is requisite for the IL-1-induced proliferative response because on the one hand the growth-promoting activity of IL-1 is completely inhibited by an anti-IL-7 mAb, and on the other hand IL-7 synergizes with IL-1 on thymocyte growth. This synergy is observed even at concentrations at which IL-7 is not detected in the pre-B cell proliferation assay, and results, at optimal doses, in TdR incorporation levels similar to those attained in response to IL-1 + IL-2. The anti-IL-7 mAb acts in a dose-dependent manner and does not affect other activities of IL-1, such as its capacity to sustain the growth of the U373 astrocytoma cell line. It is also noteworthy that this mAb does not significantly impair thymocyte growth in response to IL-2 and that the growth-promoting activity of IL-1 is not affected by neutralizing mAb against IL-2, IL-4, and IL-6. In addition, we show that the potentiating effect of granulocyte-macrophage (GM)-CSF and TNF-alpha on IL-1-induced thymocyte growth is dependent on IL-7 because i) the anti-IL-7 mAb abrogates the respective synergistic interactions and ii) both factors potentiate the proliferative response to IL-7. Finally, depletion of thymocyte suspensions for Ia+ Mac-1+ accessory cells results in a considerable decrease in IL-1- and IL-1 + GM-CSF-induced TdR uptake, whereas IL-7-induced growth remains unchanged. Taken together, these results support the notion that, in the absence of artificial comitogens, thymocyte proliferation in response to IL-1 alone or in combination with GM-CSF is dependent on accessory cell-derived IL-7.

Hematopoietic progenitors and interleukin-3-dependent cell lines synthesize histamine in response to calcium ionophore.

The calcium ionophore A23187 promotes histamine synthesis in murine bone marrow cells by increasing the expression of mRNA encoding histidine decarboxylase (HDC), the histamine-forming enzyme. The cells responsible for this biological activity copurify with hematopoietic progenitors in terms of density, light scatter characteristics, and rhodamine retention, similar to interleukin (IL) 3-induced histamine-producing cells. Yet, the effect of calcium ionophore is not mediated by IL-3. The most purified rhodamine-bright bone marrow subset contains 80% cells that respond to calcium ionophore by increased HDC mRNA expression. This high frequency makes the involvement of one particular progenitor subset in histamine synthesis unlikely. The finding that all IL-3-dependent cell lines tested so far exhibit increased histamine production and HDC mRNA expression in response to calcium influx lends further support to this notion. Cell lines requiring other growth factors or proliferating spontaneously lack this ability. Finally, it should be noted that IL-3-dependent cell lines do not produce histamine in response to their growth factor. It might, therefore, be suggested that the pathway transducing the signal for increased histamine synthesis after IL-3 receptor binding in normal hematopoietic progenitors is modified in these cell lines.

Endogenous granulocyte-macrophage colony-stimulating factor is involved in IL-1- and IL-7-induced murine thymocyte proliferation.

We have reported previously that IL-1 induces murine thymocyte proliferation in the absence of artificial comitogens, provided that the cells are cultured at high densities. In the present study, we show that, in these conditions, TdR uptake in response to IL-1 is diminished significantly by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) Abs. Indeed, a substantial production of this growth factor occurs when thymocytes are cultured in the presence of IL-1. Maximal GM-CSF levels are attained within 3 days of culture, and mRNA expression is detected after a 48-h stimulation. Both GM-CSF production and IL-1-induced thymocyte proliferation are decreased considerably by the depletion of I-A+ Mac-1+ accessory cells. Yet, addition of exogenous GM-CSF to accessory cell-depleted thymocytes does not restore the proliferative response to IL-1 alone, suggesting the implication of another accessory cell-derived mediator. Our data design IL-7 as the endogenous factor required in our culture system because: 1) GM-CSF can reverse the decrease in the proliferation after accessory cell depletion when IL-7 is provided together with IL-1, and 2) the proliferative response to IL-1 plus IL-7 is diminished as much by neutralization of GM-CSF by its specific Abs as by accessory cell removal (approximately 30%). Finally, the cells responding to IL-1 + IL-7 were identified as mature CD4-CD8-TCR+ thymocytes by the use of bromodeoxyuridine (BrdUrd), suggesting that the GM-CSF produced by thymic accessory cells in response to IL-1 participates in IL-7-dependent, intrathymic expansion of the CD4-CD8-TCR+ compartment.

Antigenic challenge of immunized mice induces endogeneous production of IL-3 that increases histamine synthesis in hematopoietic organs.

Antigenic challenge of Nippostrongylus brasiliensis-infected mice induces a striking increase in histidine decarboxylase (HDC) activity in both spleen and bone marrow cells. This enhancement takes place within 1 h after injection, with a maximum at 4 h and a return to pretreatment values 20 h later. It is associated with the appearance of IL-3 in the sera of these mice. In addition, the intracellular histamine content in both hematopoietic organs is concomitantly increased. A similar injection of worm Ag into normal mice has no significant effect. Comparable enhancement of HDC activity and intracellular histamine content with almost identical kinetics is promoted by i.v. injection of rIL-3 into normal mice. Moreover, HDC levels in infected mice are increased to the same extent in response to either specific antigen or rIL-3 injection. Taken together these results support the conclusion that antigenic challenge of immunized mice induces endogeneous IL-3 which, in turn, promotes a rapid increase in histamine synthesis in hematopoietic organs.

IL-4-producing NK T cells are biased towards IFN-gamma production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells.

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the microenvironment, NK T lymphocytes can preferentially produce either IL-4 or IFN-gamma. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4-CD8-TCRalphabeta+ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-gamma in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-gamma is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-gamma production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-gamma secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant upregulation of the capacity of NK T cells to produce IFN-gamma after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response.

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.

Thrombopoietin induces histidine decarboxylase gene expression in c-mpl transfected UT7 cells.

The leukemic cell line UT7 is endowed with both megakaryocyte and basophil differentiation potential, as judged by its capacity to respond to PMA by displaying megakaryocytic and basophilic markers and to produce histamine by neosynthesis. Herein, we addressed the question whether the biological activities characteristic of basophil differentiation were still induced when c-mpl-transfected UT7 cells received a specific megakaryocytic differentiation signal delivered by thrombopoietin (TPO). Surprisingly, we found that histamine synthesis did effectively occur in response to the growth factor. This activity was not associated with megakaryopoiesis since it was not detected in megakaryocytes generated from CD34(+) cells cultured in the presence of TPO. Comparing different c-mpl-transfected cell lines, we found that the amount of histamine generated in response to TPO correlated with their responsiveness to PMA, but not with their level of c-mpl expression, thus revealing an intrinsic basophil differentiation potential. Both PMA- and TPO-induced histamine synthesis was reduced by PKC and MEKs inhibitors, indicating that the induction occurred through a common signalling pathway.

In vitro T cell unresponsiveness following low-dose injection of anti-CD3 MoAb.

Anti-CD3 MoAb treatment is widely used as an immunosuppressive therapy. In the present study we examined the in vitro T cell response in mice having received 24 h before a single i.v. injection of 10 microgram of anti-CD3 MoAb. We found that splenocytes from these mice displayed a dramatically decreased proliferative response to the T cell mitogens concanavalin A (Con A), anti-CD3, phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) + calcium ionophore, while the effect of lipopolysaccharide (LPS) was not impaired. T cell suppression persisted for about 10 days after anti-CD3 injection, returning to normal within 15 days. The F(ab')2 fragment of anti-CD3 had no such effect, indicating the requirement for in vivo activation. At the dose used, anti-CD3 resulted neither in T cell depletion nor in down-modulation of the CD3/T cell receptor (TCR) complex. The low proliferation was also not explained by apoptosis, following secondary challenge with Con A. Splenocytes from anti-CD3-injected mice were highly responsive to IL-2, but generated little or no IL-2, IL-3, IL-4 and interferon-gamma (IFN-gamma) when exposed to Con A. Normal cytokine production could not be restored by the addition of optimal doses of IL-2 during Con A stimulation. Transforming growth factor-beta (TGF-beta) was the only cytokine whose mRNA expression was not modified in stimulated splenocytes from anti-CD3-injected mice. Furthermore, anti-TGF-beta antibodies increased Con A-induced T cell proliferation, but not cytokine production.

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement.

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.