Bone marrow-derived mast cell differentiation is strongly reduced in histidine decarboxylase knockout, histamine-free mice

Zoltán Wiener1, Márton Andrásfalvy2, Éva Pállinger3, Péter Kovács1, Csaba Szalai3, Anna Erdei2, Sára Tóth1, András Nagy4 and András Falus1,3

1 Department of Genetics, Cell and Immunobiology, Faculty of Medicine, Semmelweis University, 1089 Budapest, Hungary 2 Department of Immunology, Eotvos Lorand University, 1089 Budapest, Hungary 3 Molecular Immunology Research Group, Hungarian Academy of Sciences, 1089 Budapest, Hungary 4 Mount Sinai Hospital Research Institute, Toronto M5G 1X5, Canada

Correspondence to: A. Falus, Department of Genetics, Cell and Immunobiology, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, 1089 Budapest, Hungary; E-mail: faland{at}dgci.sote.hu
Transmitting editor: E. Möller


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mast cells are differentiated in vitro from bone marrow precursors. In this study the development of bone marrow-derived mast cells was examined from histidine decarboxylase deficient (HDC–/–) and wild-type mice in the presence of IL-3. The number of non-adherent, tryptase- and c-kit-positive mast cells in bone marrow-derived cultures of HDC–/– mice was decreased compared to that of wild-type (HDC+/+) animals, but within the tryptase- and c-kit-positive cells there was no difference in the expression intensity of both markers between the two groups. Furthermore, less serine proteases mMCP5, mMCP6 and Fc{epsilon}RI{alpha} mRNA were detected in bone marrow-derived cell cultures originating from HDC–/– mice. Antigen-provoked degranulation through high-affinity Fc{epsilon}I receptor was also lower in HDC–/– mice. The colony assays in semisolid medium yielded a significantly lower ratio of mixed colonies and higher proportion of macrophage colonies from HDC–/– mice-derived bone marrow compared to the wild-type. In the course of the differentiation of HDC–/– -derived mast cells exogenously added histamine is unable to substitute the endogenously missing histamine. Concordantly, {alpha}-fluoromethyl-histamine, the specific inhibitor of HDC, revealed only a marginal inhibition on the differentiation of tryptase-positive mast cells from wild-type mice. These findings suggest that the effect of histamine on the IL-3-dependent development of bone marrow-derived mast cell differentiation during the early period is crucial and irreplaceable.

Keywords: bone marrow, differentiation, c-kit, gene targeting, histamine, IL-3, mast cell, tryptase


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In addition to the abundantly characterized role of histamine (HA) in the pathophysiology of immediate hypersensitivity, the involvement of HA in growth and differentiation had already been recognized in 1968 by Kahlson et al. (1), showing that regeneration of injured tissues is coupled with increased HA content. HA was shown to support hematopoiesis by IL-3-induced proliferation of bone marrow stem cells (2).

Although the function of mast cells was mostly known in the allergic immune response and in local hypersensitivity, recently their importance has been shown in multiple immunological, inflammatory (3), developmental (2) and malignant (4) processes. Mast cells are the only cells which, in addition to biogenic amines, such as HA and serotonin, contain preformed tumor necrosis factor in their granules and therefore are the first responding cells for microbiological infections (5,6). Murine mast cells store HA, acidic polysaccharides (chondroitin sulfate and heparin) and different proteases of the serine protease family: chymases (mMCP1–5) and tryptase (mMCP6 and 7). The actual protease content of mast cells dynamically varies with the microenvironment (79). Beside IL-3, which is an essential component for the differentiation of mast cells, many other growth factors such as stem cell factor (SCF) are involved. For example, platelet-derived growth factor acts via an indirect, SCF-dependent mechanism (10). Nerve growth factor also has a stimulating effect on the differentiation of mast cells (11).

In contrast to the relatively well-investigated functions, triggering signals and final maturation of mast cells, hardly anything is known about the early phases of their differentiation. A promastocyte population with an expression profile of Thy1low/c-kit+ was recognized in murine fetal blood by flow cytometry (12). However, a corresponding mast cell progenitor could not be identified in adult bone marrow (9,13). The developmental relationship between mast cells and basophils has not yet been clearly resolved, but recent data seem to confirm the existence of a common progenitor cell type in bone marrow (14). Moreover, IL-3 induces HA production of a cell type in bone marrow which co-purifies with clonogenic progenitors and is suspected to be a basophil precursor (15). HA was also shown to have a role in the basophilic differentiation potential of human pluripotent cell lines (16).

Histidine decarboxylase (HDC), the only and therefore essential enzyme in HA biosynthesis, forms HA from L-histidine. HDC gene-targeted mice lacking exon 6–8, including exon 8 encoding for the pyridoxal phosphate coenzyme-binding region, produce <0.2% of HA of the wild mice (17). The HDC–/– mice are HA-free provided they are kept on a HA-free diet. Earlier we found significantly reduced peripheral (skin and peritoneal) mast cell number and very poor granulation of these cells in HDC–/– mice, as almost ‘empty’ mast cells were seen under electron microsopy.

In this study we investigated whether HA deficiency modifies the differentiation of mast cells in bone marrow-derived cultures. We found a major reduction in the number of mast cells and their precursors, and defective degranulation characteristics in HDC–/– mice-derived mast cells, in vitro.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
The strategy to generate HDC knockout mice has been described (17). Briefly, we designed the HDC targeting construct to replace a ~2.4 kb-fragment extending from the SpeI site in intron 5 to the PstI site in exon 9 with a PGK-neor cassette. The appropriate genomic DNA fragments were obtained by PCR amplification using Sv129 ES-derived DNA as a template. The target vector and cloned into the pPNT vector and the linearized construct was electroporated into R1 ES cells (18). Genomic DNA from ES cell clones resistant to selection by G418 and gancyclovir was screened by PCR using a primer set designed to amplify a 2.8-kb fragment from exon 4 (5'-CCAGCTTGCACAGAGCTGGAGATGAACATC-3') to inside the neomycin resistance gene (5'-CTGAAGAACGAGATCAGCAGCCTCTGTTCC-3'). Three PCR-positive lines were aggregated with CD1 morulae to generate germline chimeras. All the lines permitted germline transmission and two were proved to be correctly targeted at the 3' arm as well. After germline transmission the animals were kept on a CD1 outbred background.

About 3- to 4-month-old male or female wild-type and HDC–/– mice (CD1 background) were used in all experiments.

Reagents
Recombinant mouse IL-3, IL-6 and SCF were from Serotec (Oxford, UK). Allophycocyanin-labeled anti-mouse c-kit, phycoerythrin-conjugated anti-mouse Mac1 (PharMingen, San Diego, CA), anti-mouse Thy1, anti-mouse IL-3 (Serotec), biotin-conjugated anti-mouse IgM (Sigma, St Louis, MO) and streptavidin–Cy5 (Dako, Glostrup, Denmark) were used as described by the manufacturer. Anti-mouse polyclonal tryptase (a kindly gift from Dr Gunnar Pejler, Swedish University of Agricultural Sciences) was applied at a dilution of 1:50. For enzyme cytochemistry we used Z-Gly-Pro-Arg-MßNA (Bachem, Switzerland) and Fast B blue salt (Serva, Heidelberg, Germany).

Tissue culture media and supplements were purchased from Sigma. Triton X-100 and p-nitrophenyl-N-acetyl-ß-D-glucosamine were from Sigma, and DMSO was from Merck (Darmstadt, Germany). DNP11-BSA was prepared by derivatization of BSA with 2,4-dinitrobenzene sulfonic acid (Merck) was kindly provided by Dr I. Pecht (Rehovot, Israel). Murine DNP-specific IgE class A2 was grown as ascites in mice. CR1/2- and CR1-specific scFv antibodies were prepared from hybridomas as described (19).

Cell culture
The femurs of mice were flushed with FCS (PAA Laboratories, Linz, Austria), then washed twice and counted after incubation in 3% acetic acid for 10 min to lyse red blood cells. Cells were then cultured in RPMI medium supplemented with 10% FCS, 1 mM pyruvate, in the presence of 20% supernatant of Wehi3B cell line as a source of IL-3 or recombinant IL-3 (1 ng/ml), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma). The starting cell concentration was 1–1.1 x 106 cells/ml. SCF was applied at 40 ng/ml. HA content of FCS was negligible as controlled by a sensitive HA ELISA kit (Immunotech, Marseilles, France) (results not shown). HA (Sigma) was used at 10–5 M concentration and {alpha}-fluoromethyl HA ({alpha}-FMH) (Sigma) at 5 x 10–5 M.

In our experiments after culturing for 5 days non-adherent cells were transferred into a new flask and half of the medium was replaced. This procedure was repeated every 4 or 5 days. For the study of early the differentiation profile we cultured cells for a maximum of 18 days, while for the degranulation assays we used cultures with pure mast cell content (~95% mast cell content as detected by c-kit expression; cultures >4 weeks old).

Secretory response of bone marrow-derived mast cells
Degranulation of bone marrow-derived mast cells in response to stimulation by Fc{epsilon}RI clustering was monitored by measuring the activity of the granular enzyme ß-hexosaminidase. For the assay DNP-specific murine IgE mAb, A2 (3 µl ascites), was added to 106 cells in 10 µl MEM and incubated in 96-well plates (100 µl suspension/well) for 2 h. Then the cells were washed 3 times with Tyrode’s buffer and stimulated with the antigen (DNP11-BSA). Following incubation at 37°C for 1 h, 20 µl supernatants were taken and incubated with 50 µl substrate solution (1.3 mg/ml p-nitrophenyl-N-acetyl-ß-D-glucosamine in 0.1 M sodium citrate, pH 4.5) at 37°C for 45 min. The reaction was terminated by the addition of 150 µl 0.2 M glycine, pH 10.7, and the optical density of the samples was measured at 405 nm.

Semisolid colony assay
Cells were plated at a density of 1 x 105 cells/ml in 1% methylcellulose-containing medium supplemented with additional factors (see above). The number of different colonies was counted on day 14 by a phase-contrast microscope (Nikon Diaphot TMD).

Enzyme cytochemistry and alcian blue-safranin staining
For the detection of tryptase activity cells were fixed with 0.6% PFA and 0.5% acetic acid for 5 min, and then incubated in the presence of 0.5 mg/ml artificial substrate (Z-Gly-Pro-Arg-4 MßNA) and 0.5 mg/ml Fast Blue B at 37°C for 90 min. The proportion of tryptase-positive cells was then counted. For staining with alcian blue-safranin, cells were fixed in ice-cold methanol for 10 min and then incubated in the staining solution for 35 min.

Flow cytometry
Samples were labeled in PBS + 1% BSA for 20 min at room temperature and then 10,000 cells were collected by a flow cytometer (Becton Dickinson, Heidelberg, Germany). For measuring the tryptase content, samples were prior fixed with 4% PFA and permeabilized by 0.1% saponin (Sigma).

RT-PCR analysis
RNA from non-adherent cells in the cultures was isolated according to Chomczyinski (20). Reverse transcription was performed with 5 mM MgCl2, 1 mM dNTP, random hexamer primer (Promega), 50 U/reaction MuLV reverse transcriptase (Perkin Elmer) and 0.5 µg RNA in 40 µl final volume. For PCR reactions Taq polymerase was purchased from Promega, the annealing temperature was 55°C for 30 cycles. The primers used were: mMCP6, 5'-ACTGTCCCTCCTGGCTAGTC-3' and 5'-TTCACAGGGACCTCAAGCTC-3' (370 bp), mMCP5, 5'-GTTCCAGCACCAAAGCTGGAG-3' and 5'-AGTTGAAGTTG GCAGAGAGTG-3' (368 bp), Fc{epsilon}RI{alpha}, 5'-TGCCCAGCTGT GCCTAGCAC-3' and 5'-CCACCTGCCTAAGATAGCCC-3' (526 bp) G3PDH was applied as housekeeping control: 5'-CAGTATGACTCCACTCACGGC-3' and 5'-TCACGCCACA GCTTTCCAGAG-3' (452 bp).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells flushed from the bone marrow of wild-type and HDC–/– mice were differentiated in the presence of either recombinant IL-3 or the supernatant of the Wehi3B cell line. The number of non-adherent cells in the HDC–/– mice-derived bone marrow cultures was ~20–25% of that of wild-type mice after 8 days of culture and similar differences were observed during the whole time period of early (up to day 18) in vitro differentiation (data not shown). Simultaneously, the proportion of alcian blue and tryptase (mMCP6) (Fig. 1) -positive cells was also significantly decreased, detected by either flow cytometry or enzyme cytochemistry. The specificity of enzyme cytochemistry for tryptase was checked on adherent (non-mast) cells in the same cultures and on other cell lines (Wehi3B), and we could observe no signals in these cases (data not shown). Similarly, the size of the c-kit-positive cell population detected by flow cytometry was highly reduced in HDC–/– mice-derived cultures compared to those of HDC+/+ origin (Fig. 1). However, there were no significant differences in the levels of either c-kit or tryptase expression between the two groups (Fig. 1).



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Fig. 1. Proportion of tryptase-positive cells (A) and the expression level (B) of this marker (detected by flow cytometry) in HDC knockout (HDC–/–) and wild-type (HDC+/+) mice. Ten days of culture, n = 5–7. Proportion of c-kit-(CD117)-positive cells (C) and fluorescence intensity reflecting c-kit expression (D) is shown after 13 days of culture (n = 5–7). A characteristic output from c-kit detection by flow cytometry is also shown (day 9, E). In all cases cells were differentiated in Wehi3B supernatant-containing cultures. Open symbols, wild-type (HDC+/+); stripped symbols; HDC–/– (HDC knockout). *P < 0.01 (Student’s t probe); NS, not significant (P > 0.05). For details, see Methods.

 
Compared to IL-3, SCF alone resulted in very few (if any) tryptase-positive mast cells in either HDC–/– or wild-type cultures during the first 9 days of differentiation. However, when SCF and IL-3 were administered together, the number of tryptase-positive cells increased compared with IL-3 alone, suggesting a rather synergistic function of SCF in the differentiation even after 12–13 days of culture (Fig. 2). As we were unable to find any significant difference in the intensity of c-kit expression between HDC–/– and HDC+/+ groups (Fig. 1), it is unlikely that the sensitivity of wild-type or HDC–/–-derived mast cells to SCF differ from each other.



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Fig. 2. Proportion of tryptase-positive cells (detected by enzyme cytochemistry) incubated in the presence of 1 ng/ml IL-3, 40 ng/ml SCF or their combination after 9 days of culture. *P < 0.01. Symbols are as in Fig. 1; for details, see Methods.

 
These data indicate that the endogenous absence of HA due to targeted disruption of HDC gene strongly impairs the maturation of mast cells in bone marrow-derived cultures.

The same conclusion was essentially drawn when the expression of various mast cell markers by RT-PCR was investigated. Compared to wild-type cells, a markedly lower level of mMCP5, mMCP6 (tryptase) and FcR{epsilon}I{alpha} mRNA was found in HDC–/– cultures on day 10 of our experiments (Fig. 3).



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Fig. 3. mMCP6 (tryptase), mMCP5, Fc{epsilon}RI{alpha} and G3PDH mRNA analysis by RT-PCR of bone marrow-derived non-adherent cells from HDC–/– and wild-type mice after 10 days of culture in the presence of Wehi3B (as a source of IL-3) supernatants. Lanes 1, cDNA control; lane 2, wild; lane 3, HDC–/–). For details, see Methods.

 
Furthermore, to decide whether this difference between wild-type and HDC–/– cultures already developed in an earlier phase of differentiation, immunophenotyping of the promastocyte population (Thy1low/Mac1–/low/c-kit+) was performed by flow cytometry. The data show that the size of this cellular set was significantly lower in HDC–/– cultures, while the relative fraction of Mac1+/Thy1/c-kit cells (monocytic lineage) was increased (Fig. 4A). Based on this result, the difference in mature mast cell number can hardly be explained by a terminal inhibition of the maturation of these cells. To test this hypothesis colony assays in methylcellulose semisolid medium with freshly isolated bone marrow cells were performed as well. The data show that in case of HDC–/– mice-derived bone marrow cells the proportion of macrophage-like colonies was increased, but that of mixed ones was decreased (Fig. 4B).




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Fig. 4. Immunophenotypic analysis of 6-day-old liquid cultures (A) and colony formation (B) of freshly isolated bone marrow cells in semisolid medium from HDC–/– and wild-type mice. Thy1low/c-kit+/ Mac-1–/low phenotype corresponds to promastocytes, Mac-1+ cells are precursors of the monocytic/macrophage lineage. *P < 0.01. Labels are as in Fig. 1; for details, see Methods.

 
The major role of the early period is further supported by the results from experiments performed with either {alpha}-FMH (a specific and irreversible inhibitor of HDC) on bone marrow-derived mast cells from wild-type (HDC+/+) mice and by exogenous HA on cultures from HDC–/– animals. Addition of {alpha}-FMH to wild-type cultures only slightly (P > 0.1) decreased the number of tryptase-positive mast cells. Similarly, when HDC–/– cells were cultured in the presence of exogenously added HA, no appreciable enhancement was detected during the early phase of differentiation. Therefore we concluded that HA might have a very early effect on the maturation process in the bone marrow (Table 1).


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Table 1. Effect of histamine on HDC–/– and {alpha}-FMH on HDC+/+ bone marrow cells in vitro
 
Neutralizing anti-IL-3 antibody (10 µg/ml) to Wehi3B supernatant completely abolished the appearance of tryptase-positive mast cells both in wild-type and in HDC–/– cultures (not shown). However, some non-adherent cells appeared in these cultures, although at a reduced number. This confirms that the acting component of the supernatant is IL-3, which has an essential role in the differentiation of tryptase-positive mast cells and is probably not required to the development of all hematopoietic cell lineages.

The difference between the IgE-mediated degranulation of bone marrow-derived mast cells from HDC+/+ and HDC–/– mice was also investigated (Fig. 5). Despite the fact that mast cells obtained from these animals express closely identical number of Fc{epsilon}RI (Table 2), the response of the HDC–/– mast cells to receptor cross-linking with different amounts of antigen is significantly reduced. It should also be noted that the total granular enzyme content of mast cells derived from the HDC gene targeted animals was ~15–20% less than that of the cells derived from control animals.



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Fig. 5. Mastocytes were differentiated from the bone marrow cells of HDC+/+ and HDC–/– mice by culturing the cells in IL-3-containing medium for >4 weeks. Cells sensitized with DNP-specific IgE were stimulated with various amounts of antigen and the degranulation was detected by measuring the activity of ß-hexosaminidase released into the supernatants after stimulation for 1 h at 37°C. Results are presented as net percentages of the cells’ total ß-hexosaminidase activity. Total enzyme content of HDC+/+, 100%; total enzyme content of HDC–/–, 83%. n = 4. *P < 0.01. Symbols are as in Fig. 1

 

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Table 2. The expression of surface molecules on bone marrow-derived mast cells from HDC+/+ and HDC–/– mice
 
Comparing the expression of several cell membrane molecules (Table 2), no significant difference was found between HDC+/+ and HDC–/– bone marrow-derived mast cells.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One of the most striking phenotypes of HDC–/– mice is the reduced number of mast cells in the peritoneal fluid and in the skin (17). In the present study we wondered about the developmental stage of mast cells in which this difference is generated in vitro. HA, a bioactive amine with broad functional significance in physiological and pathological events, is also involved in hematopoiesis. Co-incubation of isolated bone marrow with IL-3 largely increases the expression of HDC in a cell population co-purifies with stem cells preferentially belonging to the basophilic lineage (15). In this work evidence is provided that the lack of HA in HDC–/– mice affects the composition of bone marrow and inhibits the differentiation of mast cells.

Our initial observation was that 75–80% less non-adherent cells appeared in HDC–/– mice bone marrow-derived cell cultures at the early phase of differentiation (up to day 18). When we examined these cells in respect to tryptase (mMCP6 and 7), mMCP5 or c-kit, we found that the fraction of positive cells was decreased in parallel, suggesting that mast cell maturation is inhibited. However, neither the irreversible HDC blocker {alpha}-FMH (21) given to wild-type cultures nor HA administered to HDC–/– ones was able to influence significantly the differentiation profile, so we investigated whether bone marrow is involved in this observed alteration. Colony assays in semisolid medium confirmed our findings of association of HA deficiency with retarded mast cell maturation. Simultaneously, the proportion of macrophage-like colonies was increased in semisolid cultures of HDC–/– mice origin and the size of Mac1+ population was also increased in liquid cultures. This suggests a characteristic shift of differentiation patterns toward a macrophage precursor predominance in the bone marrow of HA-deficient mice. Obviously, IL-3 cannot induce HDC in the bone marrow of HDC-deficient mice and this may explain the observed inhibition. It is possible that HA can modify the IL-3 expression or rather IL-3 sensitivity of certain cell types in the bone marrow. According to another hypothesis, mast cell lineage needs the highest level of IL-3 to start differentiation (22) and HA may influence IL-3-content of the bone marrow. We are currently testing this cytokine and cytokine receptor expression in bone marrow of wild-type and HDC-deficient mice.

In our culture conditions SCF alone was unable to support the differentiation of tryptase-positive mast cells in the first 9 days. When mast cells are derived from human cord blood, SCF can usually regulate the differentiation process on its own, which can be explained by the different stem cell composition of murine bone marrow and human cord blood, and their different maturation stages. Although most authors use IL-3 in addition to SCF for producing mast cells from murine bone marrow, some reported that SCF alone is sufficient as well. The discrepancy between their and our results can be interpreted as we investigated tryptase-positive mast cells during the first 9 days of differentiation in our experiments with SCF, so we cannot exclude that these cells began to express tryptase only in a later phase. This explanation is further supported by a recently published paper where the authors detected mMCP6 mRNA expression only after day 10 of culturing the L138.8A immature mast cell line with SCF (9).

When investigating the expression level of tryptase and c-kit (receptor for SCF), we were unable to detect a significant difference between the two groups. Based on these results it is unlikely that there is any difference in the SCF sensitivity of bone marrow cells with HDC–/– and wild-type origin.

When cultures were maintained for a longer time and an almost pure mast cell population was achieved, less ß-hexoseaminidase was released from HDC–/– mast cells after antigen-induced stimulation of the cultures. This indicates that not only the early differentiation is perturbed, but the function of appearing mast cells may also be altered in HDC–/– mice.

These shown data suggest that HA is an obligatory local factor during the early, IL-3-dependent maturation of mast cell precursors in bone marrow and influences lineage pattern.


    Abbreviations
 
{alpha}-FMH—{alpha}-fluoromethyl HA

HA—histamine

HDC—histidine decarboxylase

PDGF—platelet-derived growth factor

SCF—stem cell factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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