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In previous studies, we have shown that some, but not all low-, intermediate-, and high-purity factor VIII concentrates inhibit interleukin-2 (IL-2) secretion from phytohemagglutinin (PHA)-stimulated T lymphocytes. We now present evidence that this inhibitory action of concentrates is, at least in part, due to contamination with transforming growth factor-beta (TGF-beta). Originally identified in platelets, TGF-beta is a 25-kD homodimer that has been shown to be a natural and potent inhibitor of many immunologic responses. Using a specific bioassay, we have measured TGF-beta in various factor VIII concentrates. While some concentrates contained substantial amounts of the cytokine, there was a wide variation in concentrations of TGF-beta in different products. These levels correlated with the degree of inhibition of IL-2 secretion from T cells exhibited by each product (P = .0001). Noninhibitory concentrates contained no detectable TGF-beta. Addition of a specific TGF-beta 1 antibody reversed the inhibitory effect of some concentrates on IL-2 secretion by PHA-stimulated Jurkat T cells and interleukin-5 (IL-5)-induced proliferation of an erythroleukemic cell line. These findings suggest that TGF-beta contamination is a major contributory factor to the inhibitory activity of some factor VIII concentrates on cytokine secretion or activity, and may partially explain the reported immunosuppressive effects in recipients of these blood products.

Aim-To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines. Methods-Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry. Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli. Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-y (IFN-y) by enzyme linked inumunosorbent assay (ELISA). Results-Patients with PPP (n = 18), when compared with normal subjects (n= 10), had increased percentages of CD64 + monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%) . Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36). These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia. The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n=5). G-CSF, GM-CSF and IFN-y concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples.There was no correlation between cytokine concentrations and the expression of CDl1b, CD14, CD18, and CD64 on patients' phagocytes. Conclusions-Increased expression of CDllb and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these celis are activated in vivo in patients with PPP. The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone. (3 Clin Pathol 1995;48:525-530)

Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma-interferon (gamma-IFN), or tumor necrosis factor-alpha (TNF-alpha) triggered the rapid, stable phosphorylation of a 75-Kd protein (p75) when incubated with permeabilized HL60 human myeloid leukemia cells in the presence of [gamma-32P] ATP. Among several chemical inducers of HL60 cell differentiation, dimethyl sulfoxide also triggered p75 labeling, but retinoic acid or 12-O-tetradecanoylphorbol-13-acetate did not elicit this response. Pretreatment of cells with G-CSF or GM-CSF for more than 30 seconds before permeabilization rendered the p75 labeling undetectable, suggesting that ligand-stimulated labeling was rapidly completed within this time in intact cells. Phosphorylation of p75 occurred on serine and tyrosine residues. This conclusion was confirmed by direct phosphoamino acid analysis. Immunoblot analysis of lysates of intact HL60 cells that had been incubated with G-CSF, GM-CSF, IFN, or TNF confirmed that tyrosine phosphorylation of a p75 also occurred in response to these cytokines in intact cells. Pretreatment of intact HL60 cells with one biologic agent or dimethyl sulfoxide abolished p75 labeling in response to incubation of permeabilized cells with a second agent, strongly suggesting that the same protein was phosphorylated in response to these treatments. p75 labeling was strictly dependent on expression of the appropriate ligand receptor. Data suggest that activation of a tyrosine kinase system is an early response to the binding of G-CSF, GM-CSF, TNF, or IFN to their respective cell surface receptors, or to the addition of dimethyl sulfoxide, and that the resulting phosphorylation event(s) may play a role in securing common elements in the biologic responses to these agents.