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. 2012 Feb;30(2):222-31.
doi: 10.1002/stem.771.

Characterization and Function of Histamine Receptors in Human Bone Marrow Stromal Cells

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Free PMC article

Characterization and Function of Histamine Receptors in Human Bone Marrow Stromal Cells

Krisztian Nemeth et al. Stem Cells. .
Free PMC article

Abstract

There are several clinical trials worldwide using bone marrow stromal cells (BMSCs) as a cellular therapy to modulate immune responses in patients suffering from various inflammatory conditions. A deeper understanding of the molecular mechanisms involved in this modulatory effect could help us design better, more effective protocols to treat immune mediated diseases. In this study, we demonstrated that human BMSCs express H1, H2, and H4 histamine receptors and they respond to histamine stimulation with an increased interleukin 6 (IL-6) production both in vitro and in vivo. Using different receptor antagonists, we pinpointed the importance of the H1 histamine receptor, while Western blot analysis and application of various mitogen-activated protein kinase inhibitors highlighted the role of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase kinases in the observed effect. When BMSCs were pretreated with either histamine or degranulated human mast cells, they exhibited an enhanced IL-6-dependent antiapoptotic effect on neutrophil granulocytes. Based on these observations, it is likely that introduction of BMSCs into a histamine-rich environment (such as any allergic setting) or pretreatment of these cells with synthetic histamine could have a significant modulatory effect on the therapeutic potential of BMSCs.

Figures

Fig. 1
Fig. 1
Human BMSCs express histamine receptors Immunostaining of cultured human BMSCs using specific antibodies to recognize the H1(A), H2 (B), H3 (C) and H4 (D) receptors. The green fluorescence is due to the Alexafluor-488 secondary antibody. The bar represents 25 µm. The upper right insets demonstrate RT-PCR results using receptor specific primers. The arrows point at the expected size of the receptor proteins (all around 500 bases). The H1, H2 and H4 receptors are present with both techniques, while the H3 does not seem to be expressed.
Fig. 2
Fig. 2
Histamine stimulates IL-6 and IL-8 secretion in BMSCs The IL-6 and IL-8 concentrations of the BMSCs supernatants are dependent on the length of the treatment and concentration of histamine used, 10−5 M resulting in the largest effect when IL-6 is measured (A) and 10−4 M being the strongest stimulator when IL-8 release is studied (B). Using specific antagonists to block the effect on the individual histamine receptors demonstrates that H1 receptor block eliminates the increase in IL-6 at all concentrations (C).
Fig. 3
Fig. 3
Mast cell derived factors increase IL-6 production of BMSCs in vitro. A: IL-6 production of BMSCs is not effected by co-culturing them with human MCs at a 1:1 ratio. When the MCs are first degranulated (ActMC) and then co-cultured with the BMSCs, there is a significant increase in the IL-6 production (p<0.001). Adding a H1 receptor antagonist to the media significantly decreases the production of IL-6 by BMSCs. B: prostaglandin D2, another major MC mediator also induces BMSC derived IL-6 secretion. Since PGD2 was dissolved in ethanol, the individual controls (C1-3) contain the same EtOH concentration. In 10−6 and 10−5 Molar concentrations PDG2 significantly increases the IL-6 production of BMSCs after 6 hours of treatment.
Fig. 4
Fig. 4
Human BMSCs produce increased amounts of IL-6 in vivo in a HDC-and mast cell- dependent manner. Human BMSCs were placed into the peritoneal cavity of control and histamine-deficient (lacking histidine decarboxylase or HDC, a vital enzyme in histamine biosynthesis) mice afer inducing MC degranulation. Six hours later the increase in IL-6 content of the cell-free peritoneal wash was significantly reduced, but still present, in the histamine-deficient mice. Once again this suggests that increased IL-6 production is due to not only histamine but also other MC derived factors. When BMSCs were introduced into MC-deficient mice the same sensitisation/challenge protocol resulted in no IL-6 suggesting the MCs to be responsible for the effect.
Fig. 5
Fig. 5
Histamine stimulation of BMSCs regulates IL-6 production via the MAP Kinase Pathway (A) We measured cytosolic Ca2+ concentrations with Fura2 in individual cells. Addition of histamine (10−5 M) evoked a rapid increase in cytosolic Ca2+ peaking at 30 seconds after stimulation, followed by a sustained elevation. (B) IL-6 increase in the presence of specific PLC inhibitor (C) Western blot analysis shows the presence of p38, ERK and JNK. Addition of histamine elicited the activation of p38, ERK, and JNK after 15 minutes and declined to basal level by 60 minutes. (D) IL-6 increase in the presence of specific MAP kinase inhibitors. SB203580, a specific p38 inhibitor, PD98059, a specific ERK inhibitor and SP600125, a specific JNK inhibitor all significantly impaired histamine's ability to induce BMSCs IL-6 production.
Fig. 6
Fig. 6
Histamine prestimulation increases the IL-6 driven antiapoptotic effect of BMSCs on peripherial blood derived granulocytes. When placed in co-culture BMSCs effectively suppressed spontaneous apoptosis of neutrophils as measured by Annexin-V binding and concurrent 7-AAD staining using FACS (A-C). Histamine prestimuation of BMSCs was able to even further decrease the number of apoptotic PMNs. This potentiation of antiapoptotic function was eliminiated in the presence of neutralizing anti-IL-6 antibodies in the media. In another set of experiments instead of adding histamine itself we used degranulated human mast cells as a source of histamine to prestimulate the stromal cells. These pretreated BMSCs exhibited a significantly greater antiapoptotic effect as compared to histamine stimulation alone. Moreover this increased pro-survival phenotype was only partially suspended in the presence of H1 receptor blockade. IL-6 neutralization eliminated the anti-apoptotic effect. Neutrophil granulocytes significantly increased their fMLP induced superoxide production when they were co-cultured with BMSCs. Histamine prestimulation of BMSCs further increased the superoxide production of neutrophil granulocytes (D).
Fig. 7
Fig. 7
Number of live neutrophils in the peritoneal wash of mice with zymosan induced peritonitis. When the intraperitoneally injected BMSCs were pretreated with histamine there were almost three times more live neutrophils, than without pretreatment.
Fig. 8
Fig. 8
A schematic drawing summarizing the hypothesized effect of histamine stimulation on BMSCs. The histamine might derive from a variety of cellular sources (or might be added as a prestimulation before delivering the cells) and effects the H1 receptor of the BMSC. In response the BMSCs will increase their IL-8 production, a cytokine that is a strong chemoattractant for neutrophil granulocytes. At the same time, the increased release of IL-6 will ensure the better survival of these attracted cells by its strong anti-apoptotic function. Thus the end-result is a larger number of live, functioning neutrophil granulocytes that will normalize an inflammatory /infectious environment.

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