Human skin in organ culture. Elaboration of proteolytic enzymes in the presence and absence of exogenous growth factors.

Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.

Recombinant human interleukin-8 restores function in neutrophils from patients with myelodysplastic syndromes without stimulating myeloid progenitor cells.

Prognosis in myelodysplastic syndrome (MDS) is not only correlated closely with blast cell count in bone marrow and chromosomal abnormalities but also correlated with decreased leucocyte count and function leading to acquisition of lethal infections. Recently, clinical trials in MDS have focused on the application of haemopoietic growth factors such as G-CSF or GM-CSF, which have proven to increase neutrophil count and function. However, these cytokines carry the risk of stimulating the malignant clone, particularly in patients with increased blast cell count. Therefore, investigation of cytokines which are able to stimulate neutrophil function without the potential risk of stimulating haemopoietic progenitor cells may be relevant for MDS. As the stimulatory effect of interleukin-8 on neutrophil function is well known, we investigated whether recombinant human IL-8 is also able to improve the function of neutrophils gained from patients with MDS. Using three different techniques--the E. coli killing assay (8 patients), the production of reactive oxygen as determined by cytochrome c reduction (7 patients) and chemiluminescence (8 patients)--a significant stimulation of neutrophil function at a concentration of 10 nm IL-8 was found in all test systems. No correlation with FAB classification was evident. On the other hand, IL-8 only mildly stimulated growth of myeloid progenitor cells in bone marrow culture of healthy individuals and MDS patients. This minimal stimulation was blocked by a neutralizing antibody directed against GM-CSF, suggesting an indirect effect of IL-8 via secondary GM-CSF release. Thus, IL-8 is able in vitro to repair the functional abnormalities of neutrophils from patients with MDS but has only a marginal influence on myeloid progenitor cells.

Endogenous serum levels and surface receptor expression of GM-CSF and IL-3 in patients with myelodysplastic syndromes.

The majority of patients suffering from myelodysplastic syndromes (MDS) die of complications due to cytopenia. Clinical trials have demonstrated that in an essential number of MDS patients cytopenia can be ameliorated by exogenously supplied growth factors. We investigated endogenous serum levels of GM-CSF and IL-3 in 15 healthy individuals and 34 patients suffering from MDS. No circulating growth factors were detected in the serum of healthy controls, nor was IL-3 measurable in MDS patients. GM-CSF serum levels, however, were elevated in a significant number of patients (26.5%). Levels did not correlate with FAB classification, leukocyte count or chromosomal abnormalities. No significant differences in GM-CSF or IL-3 receptor expression were detected between healthy individuals and MDS patients. One patient with increased endogenous GM-CSF serum level and normal surface receptor expression responded to exogenously applied GM-CSF. In the light of these results, a functional alteration of growth factor receptors or disturbances of signal transduction pathways must be discussed for MDS.

Detection of soluble IL-2 receptor in the serum of patients with myelodysplastic syndromes: induction under therapy with GM-CSF.

Sera of 15 healthy controls and 33 patients suffering from myelodysplastic syndromes (MDS) were investigated for soluble interleukin-2 receptor (sIL-2R) expression with a cell-free enzyme-linked immunosorbent assay (ELISA) system (T-Cell Sciences; Cambridge, U.S.A.). The upper limit of the assay is indicated with 477 U/ml. According to the FAB classification eight refractory anaemia (RA), 15 refractory anaemia with excess of blasts (RAEB), five refractory anaemia with excess blasts in transformation (RAEBt) and five chronic myelomonocytic leukaemia (CMML) were examined. None of the patients had reported infectious episodes or been under treatment with cytotoxic agents and/or cytokines within the previous 3 months. Significant differences in sIL-2R levels between RA (median 368 U/ml). RAEB (median 675 U/ml) and RAEBt (median 971 U/ml) and between RA and CMML (median 723 U/ml) were detected. Six patients, who had been under treatment with rhGM-CSF for at least 2 weeks, demonstrated a three- to sevenfold increase of sIL-2R expression compared to pretreatment levels. In kinetic evaluation of serum samples for 24 h, the increase of sIL-2R expression begins within 4 h after subcutaneous application of GM-CSF and reaches its maximum after 12 h. Our data cannot suggest whether increased sIL-2R expression is a primary event due to involvement of lymphocytes in the malignant clone or whether it results from secondary alteration of the cytokine network. Application of GM-CSF in MDS may result in improvement of altered lymphocyte function. As GM-CSF induces sIL-2R expression, a down regulation of the immune response caused by neutralization of free IL-2 cannot be excluded.