Elke Schneider

Trends in histamine research: new functions during immune responses and hematopoiesis.

Abstract Histamine is a bioamine with multiple physiological activities. In the immune system, it not only mediates the deleterious effects accompanying allergic reactions, but it acts also in a more subtle way by modulating the T helper 1 (Th1)–Th2 balance and possibly hematopoiesis. The histamine required for Th-cell polarization is provided by mast cell or basophil degranulation, as well as being newly synthesized and immediately released by other hematopoietic cells, in response to molecules generated during the immune response. Its global effect depends on the subtype and distribution of histamine receptors on target cells. The recent discovery of a novel H 4 receptor, which is expressed preferentially on immunocompetent cells, will have important consequences for the understanding of the regulatory functions of histamine during the immune response.

Differentiation of human basophils: an overview of recent advances and pending questions.

Basophils are rare, circulating leukocytes derived from hematopoietic CD34+ progenitors. The identification of cytokines promoting their development in vitro has led to substantial advances in understanding their differentiation process. An important role could be assigned to interleukin‐3 (IL‐3), which supports the maturation of hematopoietic progenitors into basophils in vitro and in vivo. In contrast to other myeloid lineages, a specific basophil growth factor has not yet been discovered. Furthermore, it is still unclear whether basophils possess a lineage‐restricted progenitor or whether they share a common ancestor with mast cells (MC), eosinophils, or even megakaryocytes. Partial answers to these questions could be provided using in vitro culture systems or taking advantage of hematological disorders, such as chronic and acute myeloid leukemia (CML and AML), some myelodysplastic syndromes, and the very rare acute basophilic leukemia in which basophilic differentiation occurs.

The role of arginase in the immune response

Arginase activity - mainly known for its involvement in urea formation in the liver - inhibits DNA synthesis in cultured mammalian cells(1). Arginase is synthesised and released by activated macrophages mediating immunosuppression during mixed leucocyte culture(2) and may be implicated in macrophage cytotoxicity during the anti-tumor(3), anti-parasite(4) and anti-viral(5) responses. Here Elke Schneider and Michel Dy discuss these aspects of arginase activity as well as more recent indications that arginase may be involved in the cell differentiation and/or proliferation taking place during the immune responses(6,7). A lymphokine with arginase-enhancing activity has been identified. By producing ornithine, arginase provides the unique precursor of polyamines which are essential for DNA synthesis(8).

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.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1−/−) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT2A and 5-HT2B receptors toward terminal differentiation. In addition, red blood cells from 5-HT–deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1−/− animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT–deficient and normal cells. Our thorough analysis of TPH1−/− mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

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.

Histamine, Immune Cells and Autoimmunity

Histamine is one ofthe most versatile biogenic amines with multiple roles during the immune response and in allergic disorders. With four distinct G protein-coupled receptors (H1R, HER, H3R and H4R), intracellular histamine binding sites (most likely members of the cytochrome P450 family) as well as a membrane transporter (Organic Cation Transporter; OCT3) expressed in various immunocompetent cells, it can entertain a complex network of interactions. These signaling pathways are expressed differentially, depending on the stage of differentiation or activation of target cells, thus adding a further degree of complexity to the system. For this reason, published data are sometimes conflicting and varying according to the particular cell type or responses analyzed and the experimental approaches used. On the other hand, histamine is generated by several cells during the immune response, not only through release of intracellular stores in mast cells or basophils in response to IgE-dependent or -independent stimuli, but also through neosynthesis catalyzed by histidine decarboxylase (HDC) in a number ofhematopoietic cells that secrete the amine immediately without prior storage. These features enable histamine to tune the fine balance between immunity and tolerance by affecting dendritic cells, immunoregulatory cells, T-cell polarization and cytokine production, making the way for new pharmacological strategies to control immune reactivity during immune disorders, such as autoimmunity.

Hematopoietic Changes Induced by a Single Injection of Anti‐CD3 Monoclonal Antibody into Normal Mice

The present study evaluates hematopoietic modifications consecutive to in vivo treatment of mice with anti‐CD3 monoclonal antibodies (mAb). The hamster mAb 145‐2C11, administered in a single i.v. injection of 10 μg, induced the release of both interleukin 3 (IL‐3) and GM‐CSF into the circulation. IL‐3 could be detected in the serum within 1 h, attained maximal levels after 4 h and had disappeared after 24 h. Three days later, treated mice exhibited a two‐ to threefold rise in blood neutrophil levels and increased spleen cell counts. Concomitantly, the incidence of nucleated erythroid cells in these spleens increased around 10‐fold, relative to controls having received hamster Ig. At the same time point, clonogenic progenitor frequencies were 10‐fold higher in spleens from treated mice than in those from control mice. Furthermore, the responsiveness of these splenocytes to IL‐3, in terms of histamine synthesis, was enhanced. In contrast, bone marrow cell populations were only slightly affected by anti‐CD3 injection. All hematopoietic changes required multivalent crosslinking of the mAb for induction, since F(ab′)2 fragments lacked this activity. A return to normal occurred 7‐10 days after treatment. Two i.v. injections of recombinant murine IL‐3 together with recombinant murine GM‐CSF on a single day had a less pronounced effect on progenitor cell frequencies in the spleen than treatment with anti‐CD3. This difference is probably due to the amplification of growth factor‐induced hematopoiesis by the interaction with other cytokines generated in response to anti‐CD3.

Superoxide-induced deimination of arginine in hematopoietic cells.

Murine bone marrow cells can produce citrulline directly from L‐arginine without intermediate ornithine. An L‐arginine‐dependent biochemical pathway synthesizing L‐citrulline and nitrate, coupled to an effector mechanism has also been recently demonstrated in murine cytotoxic activated macrophages. We show herein that L‐citrulline synthesis in murine bone marrow cells can be induced by the generation of superoxide. It can take place in an arginine‐free medium, suggesting the implication of a superoxide‐dependent peptidyl arginine deiminase.

Fas receptor signaling is requisite for B cell differentiation.

The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas−FasL+) or gld (Fas+FasL−) genotype into Rag‐2−/− hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor‐derived myelo‐monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.