Diagnostic criteria and classification of mastocytosis: a consensus proposal

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

https://www.pnas.org/content/pnas/92/23/10560.full.pdf

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.

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Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

Inflammatory mediators of asthma: an update.

Cytokines are usually extracellular signalling proteins, usually less than 80 kD in size, and many are glycosylated. They are produced by many different cell types that are involved in cell-to-cell interactions acting through specific receptors on the surface of target cells. Cytokines usually have an effect on closely adjacent cells and therefore function in a predominantly paracrine fashion, although they may also act at a distance (endocrine) and may have effects on the cell of origin (autocrine). Cytokines may be regarded as a mechanism for cell-cell communication, and within this group may be included growth factors and cytokines with primarily chemoattractant properties (chemokines). They act on target cells to cause a wide array of cellular functions including activation, proliferation, chemotaxis, immunomodulation, release of other cytokines or mediators, growth and cell differentiation, and apoptosis. Cytokines were originally characterised (and named) according to some aspect of their functional activity that was initially discovered, but the cloning of the genes for these cytokines has now provided a better insight into their classification and grouping. It is apparent that there is a wide pleiotropy and element of redundancy in the cytokine family in that each cytokine has many overlapping functions, with each function potentially mediated by more than one cytokine.

The effect of an individual cytokine in the context of disease may not be easy to predict because it may be influenced by other cytokines released simultaneously from the same cell or from target cells following activation by the cytokine. The effects of cytokines are mediated by binding to cell surface high affinity receptors usually present in low numbers. The number of these receptors can be upregulated with cell activation, and there the effect of a cytokine may depend on the modulation of its receptors. Cytokines themselves may induce the expression of receptors which may …

https://thorax.bmj.com/content/thoraxjnl/54/9/825.full.pdf

Cytokines in asthma

Both human and mouse c-kit ligand induced differentiation of human mast cells in a long-term culture of the mononuclear cells of umbilical cord blood. Growth factor activity for human mast cells present in conditioned medium of BALB/3T3 fibroblasts was due to mouse c-kit ligand. Recombinant c-kit ligand induced differentiation and proliferation of mast cell progenitors in early stages of culture. However, apparent selective growth of mast cells by c-kit ligand in cord blood cell cultures is mainly due to the effect of the cytokine to selectively maintain survival of immature mast cells. Electron microscopic analysis indicated that human mast cells developed by c-kit ligand were similar to human mast cells in the lung and gut mucosa, while those developed in coculture of cord blood cells with Swiss albino/3T3 fibroblasts were similar to skin mast cells. This conclusion was supported by the fact that the majority of mast cells developed by c-kit ligand contained only tryptase in their granules, whereas those developed in the cocultures contained both tryptase and chymase. It was also found that mast cells developed by c-kit ligand were immature even after culture for 14 weeks. Nevertheless, these cells express Fc epsilon RI, and could be sensitized with human IgE for anti-IgE-induced release of histamine, prostaglandin D2, and leukotriene C4.

http://www.pnas.org/content/90/2/735.full.pdf

Naoki Inagaki

Papers

Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand.