A mouse GM-CSF receptor antibody attenuates neutrophilia in mice exposed to cigarette smoke.

We investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subchronic exposure model of cigarette smoke (CS)-induced inflammation using antibodies directed against GM-CSF or the GM-CSF receptor (GM-CSFR) α-chain. CS-induced mononuclear and neutrophilic inflammation following 4 days of CS exposure in BALB/c mice was assessed in bronchoalveolar lavage (BAL) fluid. An increase in mature dendritic cells (DCs) (CD11c+ and major histocompatibility complex II+) and Gr-1-high neutrophils was also observed by flow cytometric analysis of whole-lung tissue. Daily i.p. injection of 400 µg GM-CSF or GM-CSFR antibody prior to daily smoke exposure attenuated the accumulation of neutrophils within the BAL by 60%. A reduction in mature DCs was also observed. Anti-GM-CSFR antibody administration did not have an effect on the percentage of lung T-cells; however, a significant decrease in activated CD69+ CD8+ T-cells was observed. Anti-GM-CSFR antibody administration decreased the mRNA and protein expression of interleukin-12 p40 and matrix metalloproteinase 12. Taken together, intervention with this receptor antibody implicates the GM-CSF pathway as an important mediator of smoke-induced inflammation.

Oncostatin M stimulates c-Fos to bind a transcriptionally responsive AP-1 element within the tissue inhibitor of metalloproteinase-1 promoter.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) can be regulated by gp130 cytokines such as IL-6 and oncostatin M (OSM). Polymerase chain reaction deletion analysis of the murine TIMP-1 proximal promoter in chloramphenicol acetyltransferase reporter gene constructs identified an AP-1 element (-59/-53) that allows maximal responsiveness to OSM in HepG2 cells. Fos and Jun nuclear factors bound constitutively to this site as identified by supershift analysis in electrophoretic mobility shift assays, and oncostatin M (but not IL-6) induced an additional "complex 2" that contained c-Fos and JunD. OSM stimulated a rapid and transient increase in c-Fos mRNA and nuclear protein that coincided with complex 2 formation. Phorbol 13-myristate 12-acetate could also induce c-Fos but could not regulate the TIMP-1 reporter gene constructs. Transfection studies also showed that 3'-deletion of sequences downstream of the transcriptional start site (+1/+47) markedly reduced OSM -fold induction. Nuclear factors bound to SP1 and Ets sequences were detected, but were not altered upon OSM stimulation. Although OSM and IL-6 induced STAT (signal transducers and activators of transcription) factors to bind a high affinity Sis-inducible element DNA probe, binding to homologous TIMP-1 promoter sequences was not detected. Thus, OSM (but not IL-6) stimulates c-Fos, which participates in maximal activation of TIMP-1 transcription, likely in cooperation with other factors such as SP1 or as yet unidentified mechanisms involving the +1 to +47 region of the promoter.

Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.