A role for tumour necrosis factor-alpha, Fas and Fas-Ligand in marrow failure associated with myelodysplastic syndrome.

Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), Fas-Ligand (Fas-L) and TNF-alpha factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34-positive cells and on cells co-expressing HLA-DR. In addition, Fas-L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas-L mRNAs as well as mRNA for TNF-alpha, known to increase Fas expression in normal marrow, were up-regulated in patients with MDS. TNF-alpha protein and sTNF-R1 levels in marrow plasma were higher in MDS patients than in controls (P<0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF-alpha levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-T) (P=0.043). Conversely, the proportion of Fas-L-positive cells was lowest in patients with RA (P=0.037). In marrow cultures, Fas-Ig, rhuTNFR:Fc or anti-TNF-alpha antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P<0.001, P<0.003, P<0.001, respectively). These results show significant dysregulation in the expression of TNF-alpha, Fas and Fas-L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.

HLA-DR-triggered inhibition of hemopoiesis involves Fas/Fas ligand interactions and is prevented by c-kit ligand.

The function of MHC class II (HLA-DR) Ags in hemopoiesis is not well defined. Here we investigated the effect of anti-HLA-DR mAb H81.9 on human marrow cells. mAb H81.9 inhibited colony formation from purified CD34+ marrow cells in long term culture-initiating cell assays. Inhibition was prevented, however, if c-kit ligand (stem cell factor (SCF)) was added to cultures concurrently with H81.9. DNA histograms from cultured untreated marrow mononuclear cells showed 2+/-1.2% apoptotic nuclei, whereas 14.1+/-5.4% were apoptotic after 12-h exposure to mAb H81.9. The apoptotic peak was reduced to 1.2+/-0.8% when SCF was added to cultures concurrently with mAb H81.9. The addition of Fas-Ig, a fusion protein that neutralizes Fas ligand (Fas-L), also prevented mAb H81.9-induced apoptosis. As determined by terminal deoxynucleotidyl transferase assays, agonistic anti-Fas mAb also induced apoptosis (in 13+/-4% of cells), and combined treatment with anti-Fas mAb and H81.9 was additive (27% apoptotic nuclei). The extent of apoptosis induced by anti-Fas mAb was significantly reduced by SCF. After H81.9 exposure, Fas was up-regulated on CD34+ cells, and Fas-L expression was 2.5-fold higher than in controls or CD34- cells, particularly within a small cell window with low orthogonal scatter (lymphocyte gate). These findings show that HLA-DR-mediated signals inhibit hemopoiesis in human marrow by a mechanism involving Fas/Fas-L-dependent signals that are blocked by c-kit ligand. These data suggest a possible role for MHC class II molecules in the regulation of hemopoiesis.

High-affinity peptide ligands to prostate-specific antigen identified by polysome selection.

We have used the polysome-selection method to isolate peptide ligands that bind with high affinity to Prostate-Specific Antigen (PSA), an important prostate-cancer marker. Two random libraries, each encoding approximately 10(12) random peptides, were transcribed into RNA and translated in vitro. Polysomes were panned by affinity selection of the nascent peptides against immobilized PSA. Over 30% of the selected species had significant affinity for PSA; the dissociation constant of the complex formed by the best isolate with PSA was < 10(-9) M. Formation of streptavidin conjugates of selected peptides improved their affinities and, in one case, virtually eliminated non-specific binding. The polysome-selection method can be used to produce high-affinity peptide ligands of potential use in diagnostic and therapeutic procedures.

Quantitative and functional studies of impaired natural killer (NK) cells in patients with myelofibrosis, essential thrombocythemia, and polycythemia vera. I. A potential role for platelet-derived growth factor in defective NK cytotoxicity.

In view of the possibility that immunological dysfunctions may be involved in initiating or contributing to the pathogenesis of myeloproliferative disease, we investigated quantitative and functional activity of natural killer (NK) cells in patients with polycythemia vera, essential thrombocythemia and myelofibrosis, and have demonstrated, especially in myelofibrosis, a measurable cytotoxic defect in the ability of their peripheral blood mononuclear cells to efficiently kill the standard NK target, K-562. Furthermore, highly purified, FACS-sorted CD16+ lymphoid cells from patients with myelofibrosis were consistently defective in their ability to lyse K-562 targets, and could not be augmented substantially with recombinant interleukin (IL)-2. Only patients with myelofibrosis had significantly lower percentages and absolute numbers of CD16+ cells as compared to patients with essential thrombocythemia and polycythemia vera. Further experiments demonstrated that patients with myelofibrosis, although having fewer CD16+ NK cells, had a significantly increased proportion of CD16+ cells with detectable platelet-derived growth factor (PDGF) on their surface. In contrast, surface PDGF was barely detectable on CD16+ cells from patients with polycythemia vera and essential thrombocythemia, as well as from normal controls. Having previously reported that physiologic quantities of PDGF significantly inhibit human NK cell cytotoxicity, and that patients with myelofibrosis and essential thrombocythemia have significantly elevated circulating levels of plasma PDGF, we now have demonstrated that pretreatment of normal NK cells with concentrated, PDGF-containing, platelet-poor plasma from patients with these diseases significantly inhibits NK cytotoxicity. This inhibitory effect was reversed by neutralization of plasma PDGF with anti-PDGF (coupled to Sepharose resin). Both the NK defects demonstrated in this study, and the abnormal plasma PDGF results reported earlier, are most striking in myelofibrosis and least abnormal in polycythemia vera, with an intermediate degree of abnormality in essential thrombocythemia. Our new findings suggest a causal correlation between abnormal platelet release, plasma accumulation of PDGF, and the observed NK immunodeficiency in these myeloproliferative patients.

Inhibition of human natural killer cell activity by platelet-derived growth factor (PDGF). III. Membrane binding studies and differential biological effect of recombinant PDGF isoforms.

We have previously reported that platelet-derived growth factor (PDGF) substantially inhibits human natural killer (NK) cell cytotoxicity, and that NK cells possess high-affinity surface binding sites for the PDGF-AB isoform. In this communication, we present direct evidence for the presence of A-type (alpha) PDGF receptors on human NK cells by demonstrating that human NK cells have approximately 150,000 high-affinity, surface binding sites for recombinant (r)PDGF-AA and approximately 300,000 high-affinity, surface binding sites for rPDGF-BB. This was determined by the competitive binding of 125I-labelled rPDGF-AA or 125I-labelled rPDGF-BB and homologous unlabelled rPDGF-AA or rPDGF-BB to FACS-sorted, CD16+ lymphoid (NK) cells, and Scatchard analysis of these data. In addition, we also demonstrate that the various isoforms of PDGF have differential effects on NK-cell cytotoxicity. Physiological quantities (100 ng/ml) of rPDGF-BB homodimers, highly purified PDGF-AB heterodimers from outdated platelets, and rPDGF-AB heterodimers substantially inhibited NK-cell cytotoxicity in both a dose- and time-dependent manner. In contrast, pretreatment of NK cells with equivalent nanogram amounts of rPDGF-AA homodimers resulted in a significantly weaker inhibitory effect on NK-cell cytotoxicity as compared with the PDGF-BB and PDGF-AB isoforms. The implications of these findings are discussed.

Platelet-derived growth factor concentrations in platelet-poor plasma and urine from patients with myeloproliferative disorders.

Our enzyme-linked immunosorbent assay (ELISA) for measuring human platelet-derived growth factor (PDGF) detects nanogram quantities (ranging from 0.007 to 16 ng/100 microL) in purified PDGF standards. This assay is sensitive enough for studying plasma and urine. The range in normal volunteers was 0.6 to 2.3 micrograms/L for platelet-poor plasma and 1.4 to 3.3 micrograms/L for urine. We determined PDGF levels in the circulation (outside platelets) in patients with myeloproliferative diseases. Platelet-poor plasma and urine PDGF were significantly elevated in patients with myelofibrosis (6.2 +/- 2.0 micrograms/L for plasma; 7.8 +/- 2.4 micrograms/L for urine) and essential thrombocythemia (5.5 +/- 1.5 micrograms/L for plasma; 11.4 +/- 2.2 micrograms/L for urine), but not in patients with chronic myelogenous leukemia (2.1 +/- 0.4 micrograms/L for plasma; 2.8 +/- 1.2 micrograms/L for urine). Polycythemia vera produced an intermediate pattern: although plasma PDGF was within the normal range (2.1 +/- 0.2 micrograms/L), urine levels were increased (3.7 +/- 0.6 micrograms/L). These results show that PDGF is increased in the circulation in some but not all myeloproliferative diseases, and suggest that this is due to abnormal in vivo release from either megakaryocytes in the bone marrow or circulating platelets.

Inhibition of human natural killer cell activity by platelet-derived growth factor. II. Membrane binding studies, effects of recombinant IFN-alpha and IL-2, and lack of effect on T cell and antibody-dependent cellular cytotoxicity.

We have previously reported that a 2-h pre-treatment with physiologic (nanogram) quantities of the naturally occurring biologic substance, platelet-derived growth factor (PDGF), significantly inhibits human NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target K-562, as well as the single-cell binding of human NK cells to K-562 targets (i.e., conjugate formation). In this study, we have completed a more detailed and longer time course evaluation of PDGF-mediated human NK cell inhibition, and demonstrate a more marked inhibition of human NK cells at 8 to 20 h as compared to 2 h. We also show that either rIFN-alpha or IL-2 reverses the PDGF-mediated NK cell inhibition; and furthermore, that PDGF does not inhibit the lymphokine augmented NK activity of recombinant alpha-IFN or IL-2-activated human NK cells. In addition, we have further demonstrated that PDGF does not inhibit cytotoxic T cell activity as generated in the MLR, nor does PDGF significantly inhibit antibody-dependent cellular cytotoxicity. Finally, we have demonstrated that human NK cells have roughly 12,000 high-affinity, surface binding sites for PDGF, as determined by the competitive binding of 125I-labeled purified PDGF and unlabeled PDGF to B73.1+ FACS-sorted lymphoid (NK) cells, and Scatchard analysis of these data. The implications of these findings are discussed.

Correlation between allergy and persistent Epstein-Barr virus infections in chronic-active Epstein-Barr virus-infected patients.

Forty-six anti-Epstein Barr nuclear antigen-positive allergic patients, 11 of whom having clinical and laboratory evidence of chronic-active Epstein-Barr virus (CA-EBV) infections, were characterized by EBV serology, percentages of T cells, B cells, and IgE+ cells, serum levels of IgE, and allergen-induced responsiveness of lymphocytes. Results demonstrated patients with CA-EBV have significantly increased responsiveness toward specific allergens, responses toward greater numbers of allergens, numbers of IgE+ T and B cells, and levels of background DNA activity in nonstimulated lymphocytes than do subjects who suffer from allergies in the absence of the CA-EBV syndrome. Further comparison between subjects with laboratory-determined mild and moderate allergy and those with CA-EBV demonstrated a progressive increase in the serum levels of IgE as the degree of allergy increased, no difference in concentrations of T and B cells, and titers of anti-viral capsid antigen and anti-early antigen to be significantly greater in patients with CA-EBV. Statistical analysis demonstrated that patients with CA-EBV could be separated from subjects with allergies by metabolic and immunologic variables. The data suggested that allergen-induced responses may contribute to the CA-EBV syndrome.

Inhibition of human natural killer cell activity by platelet-derived growth factor.

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.