Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

The Department of Cell BiologyGM-CSF/Surfactant, NO/CytokineRegulation Central to Pulmonary DiseaseOur research program focuses on understand-ing the role and regulatory aspectsof inflammatory cytokines in pulmonarydisease. Our access to the Cleveland Clinic’sextensive patient population gives us theopportunity to study cellular and molecularprocesses involved in the development of humanpulmonary diseases. Granulocyte macrophagecolonystimulating factor (GM-CSF) regulationof surfactant homeostasisand nitric oxide (NO)regulation of cytokines aretwo areas of current interest.Pulmonary alveolarproteinosis (PAP) is a rarelung disease characterized bythe accumulation ofsurfactant material within thealveoli. GM-CSF-deficientmice develop a PAP-likesyndrome that can becorrected by exogenous GM-CSF, suggesting this factor’spivotal role for GM-CSF innormal lung homeostasis andMary Jane Thomassen, Ph.D.clearance of surfactant. Administration ofexogenous GM-CSF ameliorates lung disease in asubset of PAP patients, providing support for aGM-CSF role in human PAP. Monocytes andalveolar macrophages from PAP patients produceGM-CSF and respond to GM-CSF, indicating nointrinsic defects in the cells’ ability to produceGM-CSF or in the GM-CSF receptor. Further, allPAP patients tested have antibodies against GM-CSF in both BAL and serum. Interleukin-10 (IL-10), a pleiotropic cytokine that stimulatesantibody production, is also a potent inhibitor ofGM-CSF production from alveolar macrophages.PAP patients have less detectable GM-CSF inbronchoalveolar lavage fluids but higher levels ofIL-10 than healthy controls. IL-10 polymorphismshave been associated with increased IL-10in some autoimmune diseases. A polymorphismin the GM-CSF gene of a PAP patient has beendescribed, although the functional significancehas not been studied. Based on these data, wehypothesize that in PAP, the availability of GM-CSF is decreased by anti-GM-CSF antibodies andheightened IL-10; these events may be associatedwith polymorphisms in the IL-10/GM-CSFgenes. The long-term objective of these studiesis to delineate the role of anti-GM-CSFantibodies and IL-10 in decreasing the availabilityof GM-CSF, which is pivotal in the pathophysiologyof alveolar proteinosis. These studies,coupled with data from the GM-CSF clinicaltrial, will provide novel insights into the basicmechanisms underlying human PAP.NO is an important regulatory moleculeimplicated in both pro- and anti-inflammatoryprocesses in the lung. Abnormalities in airway NOlevels are associated with such disease states asasthma and primary pulmonary hypertension (PPH).Interestingly, alterations in inflammatory cytokineshave been reported for both of these diseases.We hypothesized that NO might be involvedin cytokine regulation by alveolar macrophages. Wehave demonstrated that NO decreases inflammatorycytokine production from human alveolar macrophages.NO may regulate cytokine gene expressionthrough effects on the transcriptionfactor nuclear factor-κB (NF-κB), whichcontrols the expression of manyinflammatory cytokine and growthfactor genes. Thus, we investigatedwhether NO affects NF-κB activation inhuman alveolar macrophages in vitro andin vivo.To study the mechanism of NOeffects on NF-κB activation, alveolarmacrophages were stimulated withlipopolysaccharide (LPS) ± a NOgeneratingcompound (DETANONOate). Results indicated that NOdecreased NF-κB activation in a dosedependentmanner. NO also preventedthe degradation of the inhibitory protein I-κB.Interference with I-κB degradation resulting infailure of NF-κB activation may be one mechanismby which NO affects cytokine gene expression.In vivo investigations were performed usingfreshly isolated alveolar macrophages from healthy(control) individuals and those with asthma and withPPH. In healthy individuals, NF-κB activation isdetected at low levels. Asthma patients with highairway NO levels showed minimal NF-κB activationwhereas asthmatics with low NO levels showedsignificantly greater NF-κB activation. In those withPPH, patients with low NO had high NF-κBactivation. These in vivo results, together with the invitro observations, support an inverse relationshipbetween NO and NF-κB activation.THE THOMASSENLABORATORYSTAFFCarol Farver, M.D.PROJECT SCIENTISTSTracey L. Bonfield, Ph.D.Baisakhi Raychaudhuri, Ph.D.POSTDOCTORAL FELLOWSDaniel A. Culver, M.D.TECHNICAL ASSOCIATESSusamma AbrahamSujata BurgessAnagha MalurADJUNCT STAFFBarbara P. Barna, Ph.D.COLLABORATORSAlejandro C. Arroliga, M.D. 1Kevin K. Brown, M.D. 2Raed Dweik, M.D. 1Serpil Erzurum, M.D. 1Stan Hazen,M.D, Ph.D. 3Mani S. Kavuru, M.D. 1Alton L. Melton, M.D. 4Herbert P. Wiedemann, M.D. 1Taolin Yi, Ph.D. 51Dept. of Pulmonary and CriticalCare Medicine, CCF2National Jewish Medical Center,Denver, CO3Dept. of Cell Biology, CCF4Dept. of Medical Subspecialties/PediatricAllergy, CCF5Dept. of Cancer Biology, CCFRaychaudhuri, B., Dweik, R., Connors, M.J., Buhrow, L., Malur, A., Drazba, J., Arroliga,A., Erzurum, S.C., Kavuru, M.S., and M.J. Thomassen (1999) Nitric oxideblocks nuclear factor-κB activation in alveolar macrophages. Am. J. Respir. CellMol. Biol. 21:311-316.Raychaudhuri, B., Fisher, C.J., Buhrow, L., Malur, A., Connors, M.J., Kavuru,M.S., and M.J. Thomassen (2000) Interleukin-10 (IL-10) mediated inhibition of inflammatorycytokine production by human alveolar macrophages. Cytokine12:1348-1355.Thomassen, M.J., Raychaudhuri, B., Malur,A., and M.S. Kavuru (2000) Pulmonaryalveolar proteinosis is a disease of decreased availability of GM-CSF rather thanan intrinsic cellular defect. Clin. Immunol. 95:85-92.Thomassen, M.J., and M.S. Kavuru (2001) Human alveolar macrophages and monocytesas a source and target for nitric oxide. Int. Immunopharmacol. 1:1479-1490.81