Mutations of serine 236-237 and tyrosine 302 residues in the human lipoxin A4 receptor intracellular domains result in sustained signaling.

Lipoxin A(4) (LXA(4)) is a potent negative modulator of the inflammatory response. The antiinflammatory activities of LXA(4), such as inhibition of agonist-induced polymorphonuclear cell (PMN) chemotaxis and upregulation of beta-2 integrins, require the expression of a G-protein-coupled, high-affinity LXA(4) receptor (LXA(4)R). We now report that stimulation of PMN with proinflammatory agonist N-formyl peptides (FMLP), calcium ionophore A(23187), or phorbol mirystate acetate (PMA) is followed by marked downregulation of LXA(4) binding (B(max) decrease of approximately 45%) and decreased activation of phospholipases A(2) (PLA(2)) and D (PLD). Elucidation of the mechanisms underlying these effects was addressed by structure-function analyses of the intracellular domains of LXA(4)R. Mutant molecule, S236/S237 --> A/G (LXA(4)R(pk)) and Y302 --> F (LXA(4)R(tk)) were obtained by site-directed mutagenesis to yield receptors lacking the putative targets for serine/threonine kinase- or tyrosine kinase-dependent phosphorylation. Expression of wild-type and mutated LXA(4)R sequences in CHO and HL-60 cells was used to examine LXA(4) ligand-receptor interactions and signal transduction events. Results indicated that cells expressing LXA(4)R(pk) or LXA(4)R(tk) displayed sustained activation of PLA(2) and PLD in contrast to the transient ones obtained with LXA(4)R(wt) (peak activation at 2-3 min). Moreover, inhibition of LXA(4)-dependent PLA(2) activity by PMA in LXA(4)R(wt) transfected CHO cells was not observed in cells expressing LXA(4)R(pk). Phosphopeptide immunoblotting revealed that the functional differences between wild-type and mutant LXA(4) receptors are accompanied by distinct changes in the receptor protein phosphorylation pattern. Further characterization of these and related LXA(4)R intracellular domains will help to better understand specific events that regulate the antiinflammatory activities of LXA(4).

A pilot study of intermittent intravenous cyclophosphamide for the treatment of systemic sclerosis associated lung disease.

OBJECTIVE: Pulmonary fibrosis, a frequent manifestation of systemic sclerosis (SSc), is considered incurable. Our aim was to assess the effect of therapy with intravenous (i.v.) cyclophosphamide on the course of pulmonary fibrosis in patients with SSc. METHODS: Five patients with SSc and clinical, laboratory, or radiographic findings of interstitial lung disease were treated with cyclophosphamide (1 g) administered i.v. monthly for 48 weeks. The dyspnea score, pulmonary function tests, arterial blood oxygen content, radiologic abnormalities, and bronchoalveolar lavage (BAL) fluid cellularity were determined before and after therapy. RESULTS: The dyspnea score decreased by 42% after 48 weeks of therapy. Forced vital capacity (FVC, percentage of predicted) increased by 7%, and carbon monoxide diffusing capacity (DLCO) decreased by 12%, but these changes were not statistically significant. Arterial blood oxygenation remained unchanged. At baseline, high resolution computed tomography of the lungs showed honeycombing, reticulonodular, or ground glass patterns in each patient examined. These radiologic abnormalities did not improve during treatment. A marked decrease in BAL fluid cell number, but not in the percentage of neutrophils, was observed after therapy. Nausea and leukopenia were frequent but mild side effects. One patient developed hemorrhagic cystitis. CONCLUSION: The results suggest that intermittent treatment with i.v. cyclophosphamide reduces the severity of dyspnea, but fails to improve FVC or DLCO, or cause resolution of radiologic abnormalities, in patients with SSc.