Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti-Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12-myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins are implicated in triggering of protein tyrosine phosphorylation, and establish a link between beta 2 integrin-dependent adhesion and the protein tyrosine kinase fgr in cell signaling.

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c⁺,CD123⁻ myeloid DC (MDC)) or immunosuppressive T cell development (CD11c⁻,CD123⁺ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-γ. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c⁺ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-β, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.

The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell.

We tested the hypothesis that human polymorphonuclear leukocytes (PMN), bearing complement receptors CR1 and CR3, might also synthesize C3, particularly when activated by LPS or cytokines. Northern blot analysis of total RNA, obtained from purified PMN stimulated overnight with LPS or cytokines (IFN-gamma, TNF-alpha, and IL-1) showed the 5.3-kb RNA transcript reported for C3 in hepatocytes and monocytes. No transcripts for C4 and factor B were detected. Time course studies of C3 mRNA expression in PMN treated with LPS or TNF-alpha demonstrated a steady increase with a plateau at 24 h that correlated with secretion of C3, determined by ELISA. In contrast, IFN-gamma and IL-1 induced a transient increase in C3 transcript with a peak around 8 h after stimulation, which was not reflected in an increased rate of C3 secretion. The content of C3 protein in PMN culture media, measured by ELISA, was about 4 ng/ml/10(7) cells after overnight stimulation with LPS or TNF-alpha. A very small amount of C3 (about 0.7 ng/ml/10(7) cells) was detected in supernatants from unstimulated and IFN-gamma- or IL-1-induced PMN. Immunoprecipitation with a polyclonal anti-human C3, followed by SDS-PAGE analysis, from [35S]methionine labeled PMN, revealed the presence in culture supernatants of three major bands at 185, 115 and 70 kDa, corresponding to pro-C3, alpha and beta chains, respectively. Analysis of [14C]methylamine incorporation and of autolytic cleavage showed that the C3 produced in tissue culture by PMN contained an intact thiolester bond. The capacity of PMN to secrete functional C3 in response to LPS and TNF-alpha might be an important mechanism of host defense at sites of inflammation.