Ten, Rosa

Abstract Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (FcεRIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets IκBα, leading to the activation of the transcription factor NF-κB. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of IκBα at Ser32/Ser36 residues. Overexpression of a dominant-negative IκBα transgene containing mutations at Ser32/Ser36 completely inhibits degradation of IκBα, NF-κB activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NFκB demonstrates that IκB kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of IκBα. Cotransfection experiments with an IKKβ negative dominant completely inhibit CD23 induced NFκB activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, IκBα degradation, and NF-κB nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates IκBα at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-κB activation and gene transcription.

Abstract Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.

The mechanisms regulating human immunodeficiency virus (HIV) persistence in human monocytes/macrophages are partially understood. Persistent HIV infection of U937 monocytic cells results in NF-kappa B activation. Whether virus-induced NF-kappa B activation is a mechanism that favors continuous viral replication in macrophages remains unknown. To further delineate the molecular mechanisms involved in the activation of NF-kappa B in HIV-infected monocytes and macrophages, we have focused on the regulation of the I kappa B molecules. First, we show that persistent HIV infection results in the activation of NF-kappa B not only in monocytic cells but also in macrophages. In HIV-infected cells, I kappa B alpha protein levels are decreased secondary to enhanced protein degradation. This parallels the increased I kappa B alpha synthesis secondary to increased I kappa B alpha gene transcription, i.e., increased RNA and transcriptional activity of its promoter-enhancer. Another protein with I kappa B function, p105, is also modified in HIV-infected cells: p105 and p50 steady-state protein levels are increased as a result of increased synthesis and proteolytic processing of p105. Transcriptional activity of p105 is also increased in infected cells and is also mediated by NF-kappa B through a specific kappa B motif. These results demonstrate the existence of a triple autoregulatory loop in monocytes and macrophages involving HIV, p105 and p50, and MAD3, with the end result of persistent NF-kappa B activation and viral persistence. Furthermore, persistent HIV infection of monocytes and macrophages provides a useful model with which to study concomitant modifications of different I kappa B molecules.

SUMMARYThe participation of eosinophils in the Spanish toxic oil syndrome (TOS) was investigated. Eosinophil infiltration and degranulation in tissues from 52 patients with the TOS were examined by immunofluorescence staining for the eosinophil granule major basic protein (MBP). Serum MBP levels were determined in sera from 323 patients. Eosinophil infiltration and degranulation were found in several tissues, especially during the acute phase of the TOS, and serum MBP was significantly elevated during all phases of the disease, suggesting that eosinophils play a role in the pathogenesis of the TOS.

Abstract While androgens clearly have significant skeletal effects, the paracrine mediators of androgen action on bone are at present unclear. Interleukin-6 (IL-6) is a candidate cytokine that is produced by osteoblastic lineage cells and promotes osteoclastogenesis and bone resorption. Here, we assessed constitutive as well as IL-1β– and tumor necrosis factor-α (TNF-α)–stimulated IL-6 mRNA expression by Northern analysis and protein secretion by immunoassay in a human androgen-responsive osteoblastic cell line (hFOB/AR-6) which contains ∼4000 androgen receptors (ARs)/nucleus. Treatment with 5α-dihydrotestosterone (DHT) dose-dependently inhibited constitutive and TNF-α/IL-1β–stimulated IL-6 mRNA steady-state levels in hFOB/AR-6 cells by 70–80% at 10−7 M. In addition, testosterone also suppressed TNF-α/IL-1β–stimulated IL-6 mRNA levels by 57%, while the adrenal androgen dehydroepiandrosterone had no effect. Of note, the specific AR antagonist, hydroxyflutamide, also inhibited IL-6 mRNA levels by 70%. Consistent with the Northern analyses, treatment with 5α-DHT, testosterone, and hydroxyflutamide also inhibited IL-6 protein production by 79%, 62%, and 71%, respectively (p < 0.001), while these agents had no effect on IL-6 soluble receptor levels. Finally, we demonstrated that hydroxyflutamide treatment of hFOB/AR-6 cells markedly inhibited the activation and binding of NF-κB (a known stimulator of IL-6 gene transcription) to its response element, thus providing a potential mechanism for its effect on IL-6 production by osteoblasts. These data are consistent with the hypothesis that suppression of osteoblast IL-6 production by androgens may mediate, at least in part, the antiresorptive effects of androgens on bone. Moreover, our findings also indicate that hydroxyflutamide, which is a known AR antagonist in most tissues, may function as a selective AR modulator for effects on IL-6 production by osteoblasts.

Human eosinophil peroxidase (EPO) was purified from eosinophil granules derived from the peripheral blood of patients with eosinophilia. The molecular mass of the H and L subunits was determined by gel filtration to be 57,000 and 11,000 daltons, respectively. The partial amino acid sequences of both subunits were used to construct oligonucleotides for the screening of several cDNA libraries, including one derived from human-induced umbilical cord mononuclear cells. A cDNA clone was isolated corresponding to EPO. The nucleotide sequence revealed an open reading frame of 2,106 bp, corresponding to a prosequence, L chain, and H chain, in this order. Comparison of the EPO nucleotide sequence with other peroxidases, such as myeloperoxidase, suggests the existence of a multigene family.

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.