Gunnar Nilsson

Phenotypic characterization of the human mast-cell line HMC-1

The cell line HMC‐1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC‐1. We also studied the potential of HMC‐1 to differentiate upon treatment with conditioned media from the human T‐cell line Mo, retinoic acid or DMSO.

Mast cell infiltration correlates with poor prognosis in Hodgkin's lymphoma

Summary. Hodgkins lymphoma (HL) is characterized by a few Hodgkin, Reed–Sternberg cells (HRS) surrounded by benign cells. We recently reported that mast cells were the predominant CD30L‐positive cells in HL tumours, and that they activate HRS in vitro through CD30L–CD30 interaction. Here, we investigated the clinical importance of mast cell infiltration in the tumours of 123 patients. Tumour specimens were stained with a mast‐cell‐specific antibody that detects tryptase. Mast cells were detected in virtually every case and increasing numbers of mast cells correlated to nodular sclerosis histology (Pu2003=u20030·008). Patients with higher mast cell infiltration had a worse relapse‐free survival (Pu2003= 0·01).

Human mast cell migration in response to members of the transforming growth factor-beta family.

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved remain largely undefined. Transforming growth factor‐β (TGF‐β) isoforms regulate numerous cellular functions, including cell growth and differentiation, formation of extracellular matrix, and the immune response. In this study we have compared the potency of different members of the TGF‐β family as human mast cell chemotaxins, and analyzed the expression of TGF‐β binding proteins on human mast cells. We were able to demonstrate that the maximal chemotactic response was attained at approximately 40 fM for the three TGF‐β isoforms, with TGF‐β3 being more effective than TGF‐β1 and TGF‐β2 at this concentration. This effect was observed in both the HMC‐1 human mast cell line and in cultured primary mast cells. In addition, TGF‐β1, TGF‐β2, and less efficiently, TGF‐β3 inhibited the proliferation of HMC‐1 cells. The migratory response is probably mediated through interaction with the TGF‐β serine/threonine type I and II receptors that were found to be expressed on the cells. No expression of TGF‐β type III receptor, endoglin, or the endothelial TGF‐β type I receptor ALK‐1 could be detected. These results provide evidence that TGF‐β isoforms are highly potent chemotaxins for human mast cells and can play an important role in the recruitment of mast cells in inflammatory reactions. J. Leukoc. Biol. 67: 350–356; 2000.

Expression of CCL5/RANTES by Hodgkin and Reed-Sternberg cells and its possible role in the recruitment of mast cells into lymphomatous tissue

HL is a malignant lymphoma characterized by a small number of malignant HRS cells among a major population of infiltrating reactive cells, e.g., lymphocytes and eosinophils. We previously reported that mast cells are present in HL‐affected lymph nodes and therein are the predominant CD30L‐expressing cells. The CD30L expressed on mast cells is functionally active and can provide stimulatory signals to HRS cells. Thus, mast cells constitute an important portion of the infiltrating reactive cells that contribute to tumor progression in HL. Control of the recruitment of this previously unrecognized cell and its interactions with tumor cells are essentially unknown. To elucidate if mast cells might be specifically attracted to the tumor area by chemokines produced by HRS cells, we investigated chemokine expression in HL cell lines and in vivo. By RNase protection assay, mRNA expression of several chemokines could be detected in the cell lines. Despite the heterogeneous expression profile exhibited by the cell lines, 4 of 5 expressed CCL5 (RANTES) mRNA. RT‐PCR and immunohistochemistry confirmed expression of CCL5 in vivo. Furthermore, secreted CCL5 was detected in conditioned media from 3 of the cell lines. In a migration assay, we found that CCL5 present in conditioned medium could induce mast cell migration. Taken together, our results suggest that CCL5 produced by HRS cells is one mechanism by which mast cells can be attracted into the tumor tissue in HL.

Chemokine Receptor Expression by Mast Cells

There is a growing interest in the role of chemokines and their receptors in the determination of mast cell tissue localization and how chemokines regulate mast cell function. At least nine chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CX3CR1, CCR1, CCR3, CCR4 and CCR5) have been described to be expressed by human mast cells of different origins. Seven chemokines (CXCL1, CXCL5, CXCL8, CXCL14, CX3CL1, CCL5 and CCL11) have been shown to act on some of these receptors and to induce mast cell migration. Mast cells have a unique expression pattern of CCR3, CXCR1 and CXCR2. These receptors are mainly expressed intracellularly on cytoplasmic membranes. Upon an allergic activation, CCR3 expression is increased on the cell surface and the cell becomes vulnerable for CCL11 treatment. Chemokines do not induce mast cell degranulation but CXCL14 causes secretion of de novo synthesized CXCL8. Because of the expression of CCR3, CCR5 and CXCR4 on mast cell progenitors, these cells are susceptible to HIV infection and mast cells might therefore be a persistent HIV reservoir in AIDS. In this review, we summarize the knowledge about chemokine receptor expression and function on mast cells.

IgE receptor‐mediated release of nerve growth factor by mast cells

It has previously been demonstrated that the level of nerve growth factor (NGF) is increased in serum from humans with allergic diseases and asthma.

The chemokine receptor CXCR4 is expressed within the mast cell lineage and its ligand stromal cell‐derived factor‐1α acts as a mast cell chemotaxin

In this study we provide evidence that the chemokine stromal cell‐derived factor‐1α (SDF‐1α) acts as a mast cell chemoattractant through interactions with its receptor CXCR4 expressed on mast cell progenitors in the blood as well as on in vitro‐developed and leukemic mast cells. We found expression of CXCR4 on cord blood‐derived mast cells (CBMC) and on the human mast cell line HMC‐1, analyzed by RNAse protection assay and flow cytometry. SDF‐1α induced intracellular calcium mobilization in HMC‐1 cells and was chemotactic for both HMC‐1 cells and CBMC. The activity of SDF‐1α was completely blocked by treating the cells with pertussis toxin, indicating the involvement of Gi‐proteins in the signaling. By applying a transwell assay we could show that SDF‐1α induces migration of a cell population in peripheral blood that is enriched for cells with the capacity to differentiate into mast cells. These findings thus suggest a mechanism by which human mast cell progenitors may be recruited from circulation into the tissue.

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene

The human mast cell line (HMC)‐1 cell line is growth‐factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC‐1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC‐1, named HMC‐1560 and HMC‐1560,816, with different phenotypes and designated by the locations of specific mutations in the c‐kit proto‐oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing™ method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c‐kit gene causing an amino acid substitution of Gly‐560 for Val. In contrast, only HMC‐1560,816 cells have the c‐kitV816 mutation found in mast cell neoplasms causing an Asp→Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3′‐kinase (PI 3‐kinase) in both variants of HMC‐1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC‐1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.

Selective CCL5/RANTES-induced mast cell migration through interactions with chemokine receptors CCR1 and CCR4

Mast cells (MCs) accumulate at sites of allergic mucosal inflammation where they act as central effector and regulatory cells. Because chemokines are of vital importance in directing inflammatory leukocytes to the sites of inflammations, we have investigated the expression and function of CC-chemokine receptor (CCR) on human MCs. Two previously unrecognized MC-chemokine receptors, CCR1 and CCR4, could be identified on cord blood-derived MCs (CBMCs). CCR1 and CCR4 expressed on CBMCs exhibited a unique response profile. Of seven CCR1 and CCR4 agonists tested, only CCL5/RANTES act as an agonist inducing chemotaxis. The migration could be partially blocked by specific antibodies against CCR1 or CCR4, while a complete inhibition was achieved when both CCR1 and CCR4 were blocked. These results demonstrate that both CCR1 and CCR4 are functional receptors on human mast cells with capacity to mediate migration towards CCL5.

Demonstration that platelet-activating factor is capable of activating mast cells and inducing a chemotactic response

Platelet‐activating factor (PAF) is generated in a variety of inflammatory conditions in which mast cells accumulate. However, little is known about the ability of PAF to influence mast cell function directly. In this study we examine the ability of PAF to activate mast cells and regulate mast cell chemotaxis. PAF was found to induce intracellular calcium mobilization and chemotactic responses in both murine and human mast cells. PAF induced transient increases in intracellular Ca2+ concentrations with a 50% effective dose of 1u2003n m and induced significant migratory responses at PAF concentrations of 1u2003n m to 1u2003µm in the human leukaemia mast cell line (HMC‐1). Using signal transduction inhibitors, both PAF‐induced calcium mobilization and migration of mast cells were shown to require activation of pertussis toxin‐sensitive G proteins. PAF‐induced calcium and chemotactic responses were cross‐desensitized by C5a. Together, these data demonstrate that PAF is capable of activating distinct signalling pathways in mast cells associated with calcium mobilization and cell migration; and that PAF may thus contribute to the regulation of mast cell responses and hyperplasia at sites of inflammation.