Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.

Mastocytosis is characterized by accumulations of mast cells in various organs (1). Most cases are indolent and confined to the skin, where discrete mast cell infiltrates are associated increased epidermal melanin, a clinical picture known as urticaria pigmentosa (UP). Other forms of mastocytosis combine UP with aggressive involvement of other organs or with haemotologic abnormalities (1-4). It is not known whether all forms of mastocytosis are true neoplasms or whether some might represent reactive hyperplasias (5-7). The c-KIT proto-oncogene encodes a type III receptor tyrosine kinase (KIT) that is critical to the development and survival of mast cells and melanocytes (8-11). The ligand for KIT (KL) can stimulate mast cell development, proliferation, and mediator release (9,12-17), as well as melanocyte proliferation and pigment production (18-20). To determine the role of c-KIT in the pathogenesis of mastocytosis, we examined tissue and cells isolated from a patient with UP and aggressive systemic mastocytosis with massive splenic involvement. We found a mutation that results in constitutive activation and expression of c-KIT in mast cells of both skin and spleen. This is the first in situ demonstration of an activation c-KIT mutation in neoplastic cells. It also demonstrates the clonal and neoplastic nature of this form of mastocytes.

Granulocyte macrophage--colony-stimulating factor (GM-CSF) decreases CD1a expression by human Langerhans cells and increases proliferation in the mixed epidermal cell-lymphocyte reaction (MELR).

Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro. To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines. The percentage of cells expressing the surface protein CD1a was determined by flow cytometry. This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value. By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures. Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a. Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation. To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured. Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells. GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines. We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.

Regulation of transgenic class II major histocompatibility genes in murine Langerhans cells.

I-E is a class II major histocompatibility complex molecule normally expressed by Langerhans cells. A series of transgenic mice were developed previously that carry E alpha d gene constructs with promoter-region deletions that cause expression of I-E by different cell types when maintained on a B6 (I-E[-]) genetic background. To study cis-acting gene sequences that regulate expression of class II proteins by Langerhans cells, we identified transgenic I-E expression by tissue immunoperoxidase staining and by epidermal cell suspension immunofluorescence cytometry. Mice with a transgene containing 1.4 kilobase pairs (kb) of flanking sequence 5' to the E alpha initiation site expressed barely detectable levels of I-E on a tiny percentage of Langerhans cells, indicating that sequences promoting Langerhans cell expression of E alpha exist between 2.0 and 1.4 kb 5' of the E alpha initiation site. Removal of an additional 170 bp of 5' flanking sequence caused near-normal levels of expression by approximately one third of epidermal Langerhans cells, which contrasts with studies that showed minimal transgene expression by splenic dendritic cells in these animals. Thus, sequences between 1.4 and 1.23 kb 5' of the E alpha initiation site decrease expression of I-E by epidermal Langerhans cells, but enable I-E expression by splenic dendritic cells. These studies identify Langerhans cell-specific regulatory sequences and genetic regions controlling major histocompatibility complex class II gene expression in Langerhans cells and splenic dendritic cells. The genetic regions identified may be particularly important because differential regulation of class II major histocompatibility complex protein synthesis by Langerhans cells and dendritic cells may be crucial to immune functions of intact animals.

Isolation, detection, and amplification of intact mRNA from dermatome strips, epidermal sheets, and sorted epidermal cells.

Three different strategies for isolating RNA from epidermal cells were compared. Starting with dermatome sections frozen or disaggregated epidermal cells purified by fluorescence activated cell sorting (FACS), RNA was isolated with a guanidinium thiocyanate technique. Specific mRNA were detected by Northern blot analysis (involucrin, keratin 5, actin), or by reverse transcription and amplification with the polymerase chain reaction (PCR), using primers specific for keratinocyte products (keratins 1 and 14) and Langerhans cells (CD1a). Messenger RNA's characteristic of Langerhans cells and of keratinocytes at different stages of differentiation were detected in dermatome and epidermal sheet preparations as well as in FACS-separated cells. The use of snap-frozen dermatome sections allows the isolation of RNA from epidermis that has undergone minimal trauma and is very close to its in vivo state, but that includes RNA from some dermal cells. Extraction of RNA from Dispase-separated sheets involves slightly more manipulation of the epidermis but provides a sample free from dermal contaminants. PCR analysis of sorted epidermal cells is both sensitive and specific, but involves still greater manipulation. This final technique, however, allows the investigation of mRNA produced by small groups of epidermal cells that are still much closer to their in vivo state than if they had been cultured. By combining these techniques it is possible to determine the baseline production of specific mRNA in the skin in vivo and to assign their production to specific groups of cells with a sensitivity and specificity greater than any approach previously described.

Malignant and normal T cells show random use of T-cell receptor alpha chain variable regions in patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T lymphocytes, most of which express alpha/beta type T-cell receptors (TCRs). The cause of CTCL is unknown, but hypotheses postulating chronic stimulation of TCRs by superantigen or by a leukemogenic virus have been proposed. Either mechanism might produce bias in the TCR variable (V) region types used by the malignant cells. To determine if TCR alpha use is restricted in CTCL, we used reverse transcription and the polymerase chain reaction to determine V alpha and V beta usage by malignant cells purified from the peripheral blood of leukemic patients with CTCL. Usage of alpha chain V region segments appeared totally random; malignant lymphocytes isolated from each of six patients used different V alpha regions. As has been previously reported, no bias was found in beta chain V region usage either. In addition to productive (in frame) TCR V region mRNAs in malignant cells from each patient, we detected non-productive (out of frame) beta chain transcripts in these cells in two of six patients, and non-productive alpha chain transcripts in five of six. Residual normal peripheral blood lymphocytes from these patients showed a random, polyclonal or oligoclonal pattern of V region usage. We conclude that there is no bias in V region usage in CTCL, making it unlikely that interactions between superantigen or virus and the TCR V regions play a role in the pathogenesis of CTCL.

Immunological phenotypes of twelve cases of nonepidermotropic cutaneous T-cell lymphoma.

The immunologic phenotypes of 12 cases of nonepidermotropic cutaneous T-cell lymphoma are reported. All cases consisted of mature peripheral T-cells. There were 9 cases of helper T-cell (Th) phenotype and 2 cases of suppressor T cell (Ts) phenotype. One case, however, expressed both the Th and Ts phenotypes. Two separate cases displayed OKT9 positivity. OKT6 positive lymphoid cells were found in the dermis during the early and late stages of the disease. We suggest that these OKT6 positive cells should represent a dermal infiltrate of Langerhans cells of immature T-cells.

Cutaneous germinal center cell-derived lymphomas. A clinical histopathological and immunopathological study of 18 cases.

Eighteen cases of cutaneous germinal center cell-derived lymphomas (CGCCL) were classified into 3 types according to Kiel classification: centrocytic lymphomas (CC), 7; centroblastic-centrocytic lymphomas (CB/CC), 9; and centroblastic lymphomas (CB), 2. The duration of the disease was 3-14 months (median 9.5 months) after the first admission. In all cases, monomorphous cutaneous nodules were found as the initial manifestation of the disease. Twelve cases of CGCCL, especially those of CB and CB/CC, initially presented with normochromic anemia, a finding which is helpful in the diagnosis of the disease. Cytomorphologically, CB tumor cells were easily identified, white CC cells were hard to distinguish from the cells of nonepidermotropic cutaneous T cell lymphoma. In many cases, however, electron microscopic examination and cytochemical stains of skin biopsy tissue imprints are useful in diagnosing CGCCL. Immunoenzyme labelling (ABC method) with monoclonal antibodies indicated that B1, K and lambda positivity are very important markers for CGCCL. Our findings also showed a higher percentage of rK type in CGCCL as compared to the Western countries.

Ca2(+)-dependent amylase secretion from pancreatic acinar cells occurs without activation of phospholipase C linked G-proteins.

We have examined the influence of guanine nucleotides on Ca2(+)-dependent amylase secretion from SLO permeabilized rat pancreatic acini. GTP gamma S (100 microM) stimulated Ca2+ dependent amylase release, decreasing the EC50 for Ca2+ from 1.4 to 0.8 microM. By contrast, GDP (1mM) and dGDP (1mM) inhibited the maximal Ca2(+)-dependent secretory response. Measurement of IP3 liberation showed that Ca2+ stimulation did not increase the activity of phospholipase C (PLC) postulated to be linked to a G-protein termed Gp; GDP and dGDP must therefore be exerting their inhibitory action via a GTP-binding protein distinct from the PLC-linked Gp.

Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis.

BACKGROUND AND METHODS: The lesions of cutaneous mastocytosis are characterized by dermal infiltrates of mast cells and may appear hyperpigmented because of the presence of increased levels of epidermal melanin. Mast-cell growth factor, the ligand for the product of the c-kit proto-oncogene, stimulates the proliferation of mast cells and increases the production of melanin by melanocytes. We therefore looked for the expression of the mast-cell growth factor gene in the skin of patients with cutaneous mastocytosis using immunohistochemical techniques and the polymerase chain reaction. RESULTS: In the skin of normal subjects and those with unrelated diseases, immunoreactive mast-cell growth factor was associated with keratinocytes and scattered dermal cells, a pattern consistent with cell-bound mast-cell growth factor. In skin samples containing lesions and in clinically normal skin from patients with mastocytosis, however, mast-cell growth factor was also found free in the dermis and in the extracellular spaces between keratinocytes, suggesting the presence of a soluble form of this protein. Messenger RNA (mRNA) that can encode soluble mast-cell growth factor was present in the skin of patients as well as in that of normal control subjects. No sequence abnormalities were detected in mRNA for mast-cell growth factor from one patient. CONCLUSIONS: The altered distribution of mast-cell growth factor in the skin of patients with cutaneous mastocytosis is consistent with abnormal production of the soluble form of this factor. This abnormality is probably due to increased proteolytic processing, since it was not explained by differences in the splicing or sequence of mast-cell growth factor mRNA in the patients. Soluble mast-cell growth factor may cause the characteristic accumulation of mast cells and the hyperpigmentation of skin found in cutaneous mastocytosis. These findings suggest that some forms of mastocytosis represent reactive hyperplasia rather than mast-cell neoplasia.