Editorial

Nitric oxide, iNOS, and in¯ammation in cystic ®brosis

D. Downey and J. S. Elborn*Adult Cystic Fibrosis Centre, Belfast City Hospital, Lisburn Road, Belfast, UK

*Correspondence to:Dr J. S. Elborn, ConsultantPhysician, Adult Cystic FibrosisCentre, Level 11, Belfast CityHospital, Belfast BT9 7AB, UK

Received: 4 August 1999

Accepted: 26 August 1999

Abstract

Nitric oxide (NO) is produced from three isoforms of nitric oxide synthase (NOS), neuronal

(nNOS), endothelial (eNOS) and inducible (iNOS). Cystic ®brosis (CF) patients have an increased

bacterial load in the airways which stimulates iNOS and therefore NO production. Upregulation of

iNOS in normal epithelial cells protects the lung from damage, but in CF cells, iNOS is not

upregulated and NO production is reduced. Reduced iNOS expression is associated with neutrophil

sequestration in the lung, thus increasing the potential damage from neutrophil proteases and

reactive oxygen species. In contrast, high concentrations of NO may augment the in¯ammatory

process in acute lung injury from sepsis. Meng et al. have shown that cystic ®brosis epithelial cells,

when stimulated by a cytokine mix and co-cultured with activated neutrophils, have reduced iNOS

expression compared to normal epithelial cells. Although iNOS expression may not accurately

re¯ect activity and NO production may arise from elsewhere, this study suggests that reduced iNOS

expression may play a part in the pathophysiological processes in cystic ®brosis.

Keywords: cystic ®brosis; iNOS; nitric oxide and neutrophils

Cystic ®brosis (CF) is the most common lethal geneticdisease, caused by defective mutations in the geneencoding the cystic ®brosis transmembrane conduc-tance regulator (CFTR) protein [1]. Reduced CFTR onapical membranes of epithelial cells [2] results inabnormal ion and ¯uid transport and a reduced abilityto kill bacteria [3]. With sub-optimal lung defence,recurrent, persistent pulmonary infections and therelease of cytotoxic neutrophil products, lung damageoccurs and pulmonary function is reduced [4]. Respira-tory failure is the most common cause of death.

Nitric oxide (NO) is involved in the regulation ofpulmonary vascular responses and plays a major part inhost defence mechanisms against infection [5]. NO isproduced by the action on L-arginine of nitric oxidesynthase (NOS), which has three isoforms. Neuronal(nNOS) and endothelial (eNOS) are constitutivelyexpressed, whereas inducible (iNOS) is produced byneutrophils, macrophages, and epithelial cells followingstimulation by an in¯ammatory milieu of bacteria, LPS,and cytokines [6]. As CF is characterized by increasednumbers of bacteria in the luminal airways, it would beexpected that iNOS expression in epithelial cells shouldbe up-regulated to produce NO and protect the lung,but a number of studies suggest that this is not the case.

Explanted bronchial epithelium in CF expresseslower levels of iNOS and mRNA than epitheliumfrom normal, emphysematous or bronchiectatic lungs,possibly due to a defect in the chain of events leadingto transcription [7]. Furthermore, nitrite production (astable end-product of NO) is reduced in CF bronchialepithelium stimulated by cytokines [8]. This is alsore¯ected in lower levels of NO in nasal air [9±11].

In the study from Meng et al. in this issue of TheJournal of Pathology, the relationship of neutrophils,iNOS expression, and epithelial cells is further

explored [12]. An epithelial cell line homozygous forDF508 CFTR and one expressing normal CFTR havebeen studied. The cells were either stimulated by acytokine mix to express iNOS, or left unstimulated.They were subsequently co-cultured with isolated andactivated peripheral blood neutrophils (although thedonor origin of the blood was unclear). Immunocyto-chemistry of the CF epithelial cells showed little iNOSexpression, whether the cells were stimulated or not, orwhether neutrophils were added. This was in contrastto normal unstimulated epithelial cells, which showed amarked increase in iNOS expression with neutrophilco-incubation and was con®rmed by RT-PCR foriNOS. This study suggests that CF epithelial cellshave reduced iNOS expression compared with normalepithelial cells when co-cultured with neutrophils orstimulated by cytokines.

Reduced iNOS expression should be re¯ected in adecreased NO concentration in exhaled breath and thishas been demonstrated in CF patients (taking intoaccount ambient room NO levels) compared with non-smoking controls [13]. In one study, sputa from patientswith CF during an infective exacerbation had anincrease in sputum nitrite/nitrate levels (NOS isoformnot de®ned) but there was no difference in exhaled NOlevels [14]. In contrast, Grasemann et al. [15] found nodifference in sputum nitrite levels during an infectiveexacerbation. This suggests that NO and its stableproducts are not useful as markers of in¯ammation.

The lack of epithelial NO may underlie the excessivetransmigration of neutrophils into the airway lumen.Inhaled NO in animal studies reduces the sequestrationof activated neutrophils into the lung by changing thedeformability and inhibiting CD18 up-regulation [16].iNOS also interferes with neutrophil±endothelial inter-actions and mice lacking iNOS have increased seques-

Journal of PathologyJ Pathol 2000; 190: 115±116.

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tration of neutrophils into the lung [17]. The reducediNOS and NO in CF lungs may provide a mechanismfor the excessive traf®cking of neutrophils into theairways. If neutrophil transmigration in CF is reduced,the potential for neutrophil protease-related lunginjury may also be reduced. In contrast, it has alsobeen suggested that iNOS de®ciency may reduce lungdamage in response to LPS [18].

Evans et al. [19] suggest that only neutrophils whichhave phagocytosed foreign material have the ability toexpress active iNOS enzyme. This protein is co-localized with myeloperoxidase in the primary granules.The NO may react with superoxide in the phagolyso-some to form peroxynitrite [20], which has bactericidalproperties that may aid neutrophil killing mechanisms.A recent study showed that NO can act synergisticallywith reactive oxygen species in vitro to kill Burkholderiacepacia, which is a major CF pathogen [21]. NOproduction is also important in killing Staphylococcusaureus and other bene®ts have been alluded to earlier[22]. These are important factors in the CF host defencemechanisms. However, the overproduction of NO byiNOS has been implicated in augmenting the in¯am-matory process in sepsis-related acute lung injury [23],asthma, bronchiectasis, and chronic obstructive pul-monary disease. [23,24]

In summary, Meng et al. have provided valuable invitro evidence for the lack of iNOS expression in CFepithelial cells stimulated by cytokines or co-culturedwith neutrophils. Co-incubation of epithelial cells withmicro-organisms was not assessed and this may be animportant factor to examine in further experiments.Previous work by these authors has shown little if anyimmunostaining for iNOS in bronchial epithelium, buta proportion of the submucosal in¯ammatory cells,mostly neutrophils, stained strongly. The detection ofiNOS does not necessarily equate with functionalactivity of iNOS, as NO production in vivo couldarise from different sources and may not be accuratelyre¯ected by in vitro cell cultures or airway secretions.Normalizing concentrations of NO at the level of thepulmonary epithelium in CF may reduce infection andin¯ammation. However, NO also has the capacityto cause tissue damage due to the production ofperoxynitrite [23], and plays a part in the pathogenesisof lung injury from in¯uenza viral pneumonia [25]. NOsupplementation as a therapy may therefore be detri-mental. Further studies are required to elucidate thebalance of in¯ammatory/antimicrobial effects of iNOSand NO in the CF lung before its therapeutic potentialcan be addressed.

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116 Editorial

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