Markers and mediators of inflammation in neonatal lung disease

Markers and Mediators of Inflammation in NeonatalLung Disease

Ali O zdemir, MD, Mark A. Brown, MD,* and Wayne J. Morgan, MD

Key words: neonatal lung inflammation; bronchopulmonary dysplasia; cytokines.

INTRODUCTION

Over the past 20 years, new therapeutic approacheshave resulted in significant improvements in the outcomeof severe respiratory disease in critically ill newborn in-fants.1 However, among the survivors chronic lung dis-ease or bronchopulmonary dysplasia (BPD) has becomea serious problem, especially in the first year of life invery low birth weight premature infants. Approximately7,000 new cases of BPD occur each year.2 After initialhospital discharge, there is a high rate of readmission (upto 60%) and subsequent death (up to 20%), mainly fromcardiorespiratory failure.3 Although survival has im-proved, advances in therapy have not significantly de-creased the incidence of BPD.1,4 Inflammation andinflammatory cytokines seem to play a pivotal rolein determining the severity of lung injury. Further under-standing of the role of inflammation in the pathogenesisand pathophysiology of BPD may lead to new preventionand/or treatment strategies.

MARKERS AND MEDIATORS OF INFLAMMATION

Cytokines

Over the last decade, our understanding of inflamma-tion and the cellular immune response has expandedenormously by the identification and characterization ofintercellular, low molecular weight (<80 kDa) regulatorypeptides, called cytokines. Today, more than 50 cyto-kines have been described, and most of them are in-volved in controlling the local and/or systemic conse-quences of injury, inflammation, hemopoiesis, and tissuerepair, including fibrosis. These immune regulatory pep-tides are mainly released from monocytes/macrophages,T lymphocytes and various other cell types (B lympho-cytes, NK cells, leukocytes, fibroblasts, endothelial cells,etc.) in response to endotoxin, cellular injury, hypoxia or

hyperoxia.5–8 Generally, their production is transient andthe radius of action limited. Their effects start by bindingto specific high-affinity cell surface receptors that causealteration of gene expression in the target cells.9

The most important function of cytokines appears tobe in the local networking of intercellular communica-tion by modulating the behavior of adjacent cells (para-crine effect; e.g., chemotactic effect of IL-8 for mono-cytes and neutrophils), or affecting the cells that secretethem (autocrine effect; e.g., stimulatory effect of IL-1 onIL-2 production, which in turn causes T lymphocyte pro-liferation). In some instances, they may influence cells indistant tissues (endocrine effect; e.g., role of TNF, IL-1,and IL-6 in systemic acute phase response).10 Anothercharacteristic feature of cytokines is their ability to regu-late their own synthesis, either in a stimulatory manner(IL-1 induces TNF production and vice versa) or inhibi-tory fashion (downregulatory effect of IL-4 or IL-6 onthe production of TNF or IL-1). They may also havemodulatory actions on receptor expression, either stimu-latory (IFN-g increases the expression of TNF receptor)or inhibitory (IL-1 decreases the expression of TNF re-ceptor). In addition, specific endogenous receptor an-tagonists may block or reduce the effect of the samecytokine (IL-1 receptor blockage by IL-1Ra).11,12

The role of cytokines in inflammation and tissue injuryhas importance in the acute phase and in tissue remod-

Pediatric Pulmonary Section, Arizona Respiratory Sciences Center,University of Arizona, Tucson, Arizona.

*Correspondence to: Dr. M.A. Brown, Division of Pediatric Pul-monology, Respiratory Sciences Center, University of Arizona Col-lege of Medicine, 1501 N. Campbell Ave., Tucson, AZ 85724-5073.E-mail: [email protected]

Received 14 August 1996; accepted 15 December 1996.

Pediatric Pulmonology 23:292–306 (1997)

State of the Art

© 1997 Wiley-Liss, Inc.

eling. Feedback mechanisms and controlled secretion ofcytokines are vitally important after the initial inflamma-tory response that determines the role of inflammation inthe healing process is over. Otherwise, acute-phase pro-inflammatory cytokines (TNF, IL-1, IL-6) along withother cytokines, such as TGF-b, PDGF, and GM-CSFmay amplify the inflammatory response.13 This may di-rectly modify matrix gene expression of resident tissuecells, and thereby stimulate fibroblast recruitment andproliferation, alter collagen synthesis, and lead to subse-quent tissue fibrosis. Table 1 summarizes the major cy-tokines, their cell sources, and their roles in acute andchronic inflammation and injury.

Other Markers and Mediators

The role of lipid mediators such as the leukotrienes,PAF, thromboxane A2, and PGs in inflammation (Fig. 1)has been extensively studied and reported.14,15 Theirrapid production at the onset of inflammation results inneutrophil chemotaxis, increase in vascular permeability,albumin leakage, edema formation, and platelet aggrega-tion. Several investigators have compared the chemotac-tic activity of IL-8, LTB4, and PAF on PMN. IL-8 wasfound to be a more potent chemoattractant for PMN thanLTB4 and/or PAF, but was not as potent when comparedto their combined effect.16 IL-8 is also a potent inducer ofLTB4 production by neutrophils, thereby amplifying thechemotactic effect of IL-8 itself.17 McColl et al.18 dem-onstrated that preincubation of peripheral blood neutro-

phils with GM-CSF enhances the ability of PAF tostimulate leukotriene synthesis by increasing both ara-chidonic acid concentration and 5-lipoxygenase activa-tion. In another study, local PAF production was ampli-fied by IL-1b in a rat model of acute immune complexalveolitis.19 Today, even though there are limited com-parative studies, it seems reasonable to speculate thatcytokines and lipid mediators have both upregulatory anddownregulatory effects when acting in concert with eachother in the regulation of inflammation.

An activated PMN product, elastase (a major serineprotease), is responsible for much of the tissue damageduring the inflammatory process.20 SLPI is synthesizedand secreted by nonciliated respiratory epithelial cells,and is a potent inhibitor of neutrophil-derived proteolyticenzymes, such as elastase and cathepsin G.21,22The bal-ance between proteolytic and antiproteolytic activity isan important determinant of tissue damage. Experimentalanimal and adult human studies have demonstrated thateither an increase in release of proteolytic enzymes (elas-tase, collagenase, cathepsin G) or a decrease in antipro-teolytic activity (a1-proteinase inhibitor) leads to de-struction of the elastin and collagen framework of lungtissue.23–25In addition, a recent study demonstrated thatelastase might indirectly increase the recruitment of neu-trophils by increasing the transcription of IL-8.26 In con-trast, SLPI transcription was not modulated by IL-8, andwas only slightly responsive to high levels of TNF-a.

Fibronectin is a nonimmune opsonin that exists in aninsoluble form on most cells (fibroblasts, macrophages,epithelial cells) and in a soluble form in plasma andinterstitial fluid.27 In response to injury, it promotes theclearance of fibrin, platelets, immune complexes, andcollagenous debris by phagocytic cells during the healingphase of inflammation, but may also—when produced inexcess—contribute to tissue fibrosis.28–30It functions asa chemoattractant for phagocytic cells directly (i.e., mac-rophages), or by increasing the PMN response.28 Fibro-nectin also promotes phagocytosis indirectly by alteringthe affinity, number, or distribution of complement re-ceptors (C3b, C3bi, C1q) on the surface of phagocytes.

A recently identified family of low molecular weight(<10 kDa) cytokines appears to have both proinflamma-tory and reparative effects.31 Because of their chemotac-tic properties and the presence of cysteine amino acids intheir molecular structure, these cytokines (listed in Table2) have been designated as the C−C chemokine family.Potent chemokinetic and chemoattractant properties forinflammatory cells (mainly neutrophils, monocytes/macrophages, T lymphocytes), modulation of cytokineproduction (TNF, IL-1, IL-6) and adhesion molecule ex-pression, and enhancement of mononuclear cell prolif-eration have been ascribed to this family of molecules.32

Abbreviations

AP-1 Activator protein-1BPD Bronchopulmonary dysplasiaDNA Deoxyribonucleic acidGM-CSF Granulocyte-monocyte colony-stimulating factorGR Glucocorticoid receptorGRE Glucocorticoid response elementIFNg Interferon-gIkba Inhibitory protein kappa BaIL InterleukinIL-1RA Interleukin-1 receptor antagonistLTB4 Leukotriene B4MIP-1a Macrophage inflammatory protein-1aMIP-2 Macrophage inflammatory protein 2mRNA Messenger ribonucleic acidNF-kB Nuclear factor kappa BPAF Platelet-activating factorPDGF Platelet-derived growth factorPG ProstaglandinPMN Polymorphonuclear leukocytesRDS Respiratory distress syndromerh-SOD Recombinant human superoxide dismutaseSLPI Secretory leukocyte proteinase inhibitorsICAM-1 Soluble intercellular adhesion molecule 1TGF-b Transforming growth factorbTNF Tumor necrosis factorTRH Thyrotropin-releasing hormone

Inflammation in Neonatal Lung Disease 293

TABLE 1— Major Inflammatory Cytokines*

Cytokine Abbreviation Primary cell source(s) Biologic effects

Interleukin-1aand

Interleukin-1b

IL-1a

IL-1b

Monocytes/macrophages,T and B lymphocytes

Important immunoregulators; synergize with other cytokines topromote B and T lymphocyte proliferation; increase Il-2receptor expression; activates NK cells; induce cytokine geneexpression; activate endothelial cells; induce cyclooxygenaseand lipoxygenase gene expression; induce acute and chronicphase response; act as an endogenous pyrogen

Interleukin-2 IL-2 T lymphocytes Stimulates proliferation and differentiation of T lymphocytes;increases cytolytic activity of NK cells; promotes developmentof lymphokine-activated killer (LAK) cells; stimulatesproliferation and immunoglobulin (Ig) secretion byB lymphocytes

Interleukin-4 IL-4 Th cells, macrophages,mast cells,B lymphocytes

Induces differentiation of CD4 T lymphocytes into Th2 cells andsuppresses Th1 development; stimulates proliferation anddifferentiation of B lymphocytes; downregulates IL-1, IL-6,IL-8, TNF-a, and expression of class II MHC antigens; role inmodulation of fibrosis

Interleukin-6 IL-6 Monocytes/macrophages,fibroblasts, endothelialcells, T lymphocytes

Growth factor for B lymphocytes and polyclonal Ig production;stimulates production of acute phase proteins by hepatocytes;acts as an endogenous pyrogen

Interleukin-10 IL-10 T and B lymphcytes,epithelial cells

Supresses functional activity of macrophages; downregulatesIL-1, IL-6, IL-8, TNF-a, and expression of class II MHCantigens by monocytes and macrophages; increases Blymphocyte proliferation and Ig secretion

Interleukin-12 IL-12 Macrophages,B lymphocytes

Enhances cytolytic activity of cytotoxic T lymphocytes, NKcells, LAK cells, and macrophages; increases proliferation ofactivated NK cells and T lymphocytes; induces production ofIFN-g by T lymphocytes

Interleukin-13 IL-13 T lymphocytes Stimulates B lymphocyte growth and differentiation; inhibitsproinflammatory cytokine production by monocytes/macrophages

Interleukin-14 IL-14 T lymphocytes Stimulates proliferation of activated B lymphocytes, but notresting B lymphocytes; inhibits Ig secretion formitogen-activated B lymphocytes

Interleukin-15 IL-15 Monocytes,T lymphocytes, bonemarrow stromal cells

Stimulates proliferation of T lymphocytes; induces developmentof LAK cells; biologic activity is similar to that of IL-2

Tumor necrosis factor-aand

Tumor necrosis factor-b

TNF-a

TNF-b

Monocytes/macrophages,B and T lymphocytes,keratinocytes, NK cells,neutrophils, endothelialcells

Important immunoregulators; except for T lymphocyteproduction of IL-2 and stimulation of B lymphocytes, TNFsubserves the same or very similar functions as IL-1; generatecytolytic T lymphocytes and enhance the expression of class IIMHC antigens

Interferon-g IFN-g T lymphocytes, NK cells Increases expression of MHC class I and II antigens; enhancesphagocytic cell function

Transforming growthfactor-b

TGF-b Chondrocytes, osteoblasts,osteoclasts, fibroblasts,monocytes/macrophages,subsets ofT lymphocytes, platelets

Stimulates osteoblasts, inhibits osteoclasts; stimulates formationof extra cellular matrix; inhibits NK cell activity; inhibitsproliferation of B and T lymphocytes; promotes IgAproduction by B lymphocytes; chemoattractant for monocytes;regulates resolution of acute inflammation: inhibitsproliferation of many cell types; important role in tissue repairand fibrosis

Platelet-derived growthfactor

PDGF Platelets, endothelial cells,fibroblasts,monocytes/macrophages,smooth muscle cells

Potent mitogen for fibroblasts, smooth muscle cells, epithelialcells and endothelial cells; chemotactic factor for fibroblasts,smooth muscle cells, neutrophils, and monocytes; inhibitsNK cell activity, stimulates degranulation by neutrophils andmonocytes; stimulates collagen synthesis

Granulocyte-monocytecolony-stimulating factor

GM-CSF T lymphocytes, monocytes,fibroblasts, endothelialcells

Promotes growth and differentiation of multipotentialhemopoietic growth; stimulates physiologic activity ofneutrophils, monocytes/macrophages, eosinophils; promotesnormal surfactant turnover; role in chronic inflammation andfibrosis

*Modified with permission from Liles WC, Van Voorhis WC. Review: Nomenclature and biologic significance of cytokines involved ininflammation and host immune response. J Infect Dis. 1995;172:1573–1580. © 1995 by The University of Chicago.

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TABLE 2—Chemokines*

Chemokine Abbreviation Primary cell source(s) Biologic effects

Interferon-inducibleprotein-10

IP-10 T lymphocytes, monocytes,endothelial cells,keratinocytes

Chemoattractant for monocytes and T lymphocytes; promotesT lymphocyte adherence to endothelial cells

Interleukin-8 IL-8 Monocytes/macrophages,T lymphocytes,neutrophils, fibroblasts,keratinocytes,hepatocytes, endothelialcells, epithelial cells,chondrocytes

Chemotactic factor for neutrophils, T lymphocyte subsets,and basophils; activates neutrophils to release lysosomalenzymes, undergo a respiratory burst, and degranulate;increases adherence of neutrophils to endothelial cells;induces leukotriene B4 from neutrophils; increasesadherence of monocytes to endothelial cells

Lymphotactin T lymphocytes Chemotactic factor for T lymphocytesMelanoma

growth-stimulatingactivity

GROa/MGSA Fibroblasts, chondrocytes,epithelial cells,monocytes/macrophages,neutrophils, platelets

Chemotactic for neutrophils; activates neutrophils; stimulatesproliferation of melanoma cells

Monocyte chemotacticprotein-1/monocytechemotactic andactivating factor

MCP-1/MCAF Monocytes/macrophages,fibroblasts,B lymphocytes,endothelial cells,keratinocytes, smoothmuscle cells

Chemotactic factor for monocytes; stimulates histaminrelease from basophils; regulates cytokine production inmonocytes; stimulates IL-1 and IL-6 production; inducesadhesion molecule expression

Macrophage inflammatoryprotein-1a

MIP-1a T lymphocytes,B lymphocytes,monocytes, mast cells,fibroblasts

Chemotactic factor for monocytes, T lymphocytes, andeosinophils; chemokinetic for neutrophils; inhibitsproliferation of early hemopoietic stem cells; stimulatesIL-1, IL-6 and TNF production; functions as anendogenous pyrogen

Macrophage inflammatoryprotein-1b

MIP-1b T lymphocytes,B lymphocytes,monocytes, mast cells,fibroblasts

Chemotactic factor for monocytes and T lymphocytes;stimulates adhesion of T lymphocytes to endothelial cells

RANTES T lymphocytes, platelets,renal epithelial andmesengial cells

Chemotactic factor for monocytes, T lymphocytes,eosinophils, and basophils; stimulates histamine releasefrom basophils

*Modified with permission from Liles WC, Van Vorrhis WC. Review: Nomenclature and biologic significance of cytokines involved ininflammation and host immune response. J Infect Dis. 1995;172:1573–1580. © 1995 by The University of Chicago.

Fig. 1. Metabolic pathways ofvarious lipid mediators and theirfunction in the inflammatory pro-cess. HETE, hydroxyeicosatet-raenoic acid; HHT, heptadacatrie-noic acid; HPETE, hydroxyper-oxyeicosatraeinoic acid; LT,leukotriene; PG, prostaglandin.(Modified with permission fromAvery GB, Fletcher MA, MacDon-ald MG, eds. Neonatalogy: Patho-physiology and Management ofthe Newborn. Philadelphia: J.B.Lippincott, 1994:457pp.)


NEONATAL LUNG INFLAMMATION

Our understanding of pulmonary defense mechanismsin human infants is limited and is largely extrapolatedfrom animal studies.33 It appears that most aspects ofupper airway defense are intact at birth in term infants.Aerodynamic filtration in the conducting airways, secre-tory behavior of the airway epithelium, and airway mu-cus composition seem to be normal during the first weeksof life in term and preterm infants. There may be subtledifferences due to secretory responses in premature in-fants. However, the capacity of the immune system at thealveolar level is deficient in many aspects in both termand—especially—preterm infants. Decreased phagocyticactivity in PMNs, monocytes, and macrophages, dimin-ished humoral immunity (low immunoglobulin levels,except normal IgG levels in term infants), low concen-trations of complement and decreased expression ofcomplement receptors, and possible defects in T lympho-cyte immunoregulation in newborn infants increase therisk of lung injury from a variety of exogenous stimuli.34

Infectious agents, barotrauma and oxygen toxicity asso-ciated with mechanical ventilation, and hypoxemia arethought to be the major causes of neonatal lung injuryand inflammation.2,35,36Infants born at an early and im-mature gestational age have additional risks for lung in-jury because of severe lung immaturity and an impairedimmune system, insufficient surfactant content, and de-creased levels of antioxidant enzymes (superoxide dis-mutase, catalase, glutathione peroxidase) compared toterm infants.37–39 Furthermore, important structuralchanges in the developing lung (i.e., appearance of thecapillary network, histologically distinguishable alveoli,marked increase in the gas exchange surface area, anddevelopment of type II pneumocytes) occur from 28weeks through 40 weeks of postconceptional age.40 Ex-trauterine existence during this period is dangerous forpremature infants.

Regardless of the triggering factor, the same series ofcoordinated events appear to occur in the lungs of new-borns. During the acute phase of lung injury, a variety ofdirect or indirect stimuli (pneumonia/sepsis, trauma, hyp-oxia, hyperoxia) initiate a host response leading to pul-monary inflammation. Cytokines and other mediators ofinflammation seem to play an important role in the regu-lation and maintenance of the inflammatory response.Extensive release of proinflammatory cytokines (TNF,IL-1, IL-6), chemokines (IL-8, MIP-2), lipid mediators(LTB4, PAF, PGs), platelet factor 4, and PDGF mainlyfrom mononuclear phagocytes, platelets, and variousresident lung cells (e.g., airway epithelial cells, endothe-lial cells), as well as complement activation (C3a, C5a),cause changes in vascular permeability.31,34,41–50Thisleads to plasma protein leakage, increased adhesion mol-ecule expression by endothelial cells, and subsequent

movement of leukocytes from the pulmonary vascularcompartment to the interstitial and alveolar spaces. In thefirst hour, macrophages are the predominant cell type inthe alveolus, followed by neutrophilic exudation at 6 to12 hours, and monocytic and lymphocytic infiltration at24 to 48 hours.31 Direct cell-to-cell contact betweenstimulated monocytes/macrophages, T lymphocytes, andother cells leads to further production of proinflamma-tory cytokines and other inflammatory mediators, result-ing in a vicious circle of inflammatory stimulation.31

Additionally, activated neutrophils release mediators,such as reactive oxygen metabolites, elastase, and colla-genase, that may directly destroy the elastin and collagenframework of lung tissue.20 After the initial injury, aresolution or recovery period occurs, which peaks at day3 and returns the lung to almost normal by day 7.51

Alveolar macrophages are the prominent cells in thisphase, playing a scavenger role by removing cell debrisand plasma components.29 They may also producegrowth factors (a term largely replaced by ‘‘cytokines’’)that are important in rebuilding the normal lung struc-ture; the cytokines involved are TGF-a and TGF-b,PDGF, insulin-like growth factor I, proinflammatory cy-tokines (TNF-a, IL-1, IL-6), fibronectin, bombesin, andothers.

Feedback mechanisms between inflammatory cellsand their mediators are of vital importance in the reso-lution period, slowing the pace of the inflammatory pro-cess and allowing healing to occur. Suppression of theproinflammatory activities of macrophages, neutrophils,lymphocytes, and their respective inflammatory media-tors is brought about by antiinflammatory cytokines (e.g.,IL-4 and IL-10), antagonism between cytokines (e.g.,TNF and IL-6), cytokine receptor antagonists (e.g., IL-1Ra),a1-proteinase inhibitor response (to neutralize pro-teolytic enzyme activity), and other suppressive media-tors (e.g., prostaglandin E2).12,52–55 Alterations in thebalance of the complex network of inflammatory re-sponse at this stage normally changes the role of inflam-mation into a healing or reparative process. However, ifthe acute cycle of injury continues with further accumu-lation of inflammatory cells and production of mediators,the same process may lead to irreversible lung destruc-tion and fibrosis. Prolonged barotrauma and oxygen tox-icity from mechanical ventilation or ongoing pneumoniaand sepsis are common risk factors for chronic lung dis-ease in neonates and may amplify lung inflammation.Inflammatory cell infiltrates, predominantly alveolarmacrophages and lymphocytes, fibroblast proliferation,basement membrane thickening, and disordered collagenmetabolism are the characteristic pathologic features ofchronic lung inflammation.51 Initially, elevated inflam-matory cytokines and other mediators of inflammationare also present at this stage. However, other cytokines,TGF-b, GM-CSF, and PDGF, and fibronectin appear to

296 Ozdemir et al.

play a critical role in the overall matrix distortion, fibro-blast proliferation, collagen synthesis and alteration ofcell structures.30,56 This period usually results in exten-sive pulmonary tissue injury, interstitial and intraalveolarfibrosis, and long-lasting impairment of the architecturaland functional development of the lung. A schematicrepresentation of lung injury and the inflammatory cas-cade is summarized in Figure 2.

ROLE OF INFLAMMATION INBRONCHOPULMONARY DYSPLASIA

BPD is an important complication among low birthweight premature and critically ill infants who need in-tensive care during the first month of life. Despite ex-tensive research since the original description by North-way et al. in 1967,57 the exact mechanisms causing BPDare not fully understood. The etiology has been attributedto a number of factors: hyperoxia and barotrauma frommechanical ventilation, infection, primary surfactant de-ficiency or nutritional factors (vitamins A and/or E defi-ciency).2,58,59Regardless of the etiologic causes, recentstudies have demonstrated early and prolonged postnatalinflammation in the lungs of infants who subsequentlydeveloped BPD.

During the first days of postnatal life, influx of neu-trophils and macrophages, markers of neutrophil recruit-ment (IL-8, LTB4, PAF, complement component C5-derived anaphylatoxin, sICAM-1), proinflammatory cy-tokines (IL-1b, IL-6), increased proteolytic activity ofelastase, and markers of chronic lung injury (TGF-b,

fibronectin) are seen in lung lavage fluids of infants whosubsequently develop BPD.30,60–76Elevated levels of in-flammatory cells (neutrophils, macrophages) and elastaseactivity were observed beginning at birth until 5 weeks ofpostnatal age.60–62Also, sICAM-1, an important ligand/receptor for CD11b/CD18 that plays a role in the media-tion of neutrophil-dependent inflammation, was found tobe elevated in aspirated tracheal fluid, peaking at be-tween 1 and 2 weeks of age in patients who subsequentlydeveloped BPD.64–66

There are few studies examining cytokine levels inlung lavage fluids early in life of infants with BPD (Table3). In general, initially increased levels of proinflamma-tory cytokines (IL-1, IL-6, IL-8) are observed at differentpostnatal ages, as early as day 1, peaking usually at days10 to 14, and remaining elevated until 3 to 5 weeks ofage.65–74Bagchi et al.67 found an increase in IL-6 in lunglavage fluids for the first 2 weeks, but not for TNF-a.TNF-a levels subsequently rose in BPD patients, withthe highest levels occurring from day 14 to 28, when IL-6activity decreased. Jones et al.77 observed low levels ofthe antiinflammatory cytokine IL-10, in trachael fluidsamples from preterm infants with RDS, but not in terminfants with meconium aspiration syndrome. They spec-ulated that a limited capacity for IL-10 production mightbe a risk factor for BPD. A recent study by Kotecha etal.76 demonstrated an increase in both total and activeTNF-b levels (implicated as an important mediator offibrosis), peaking at postnatal day 4, and decreasinggradually during a 4-week period in tracheal lavagesamples from infants with BPD. Increased numbers of

Fig. 2. Schematic summary ofneonatal lung injury. GM-CSF,granulocyte-monocyte colony-stimulating factor; ICAM-1, inter-cellular adhesion molecule-1; IL,interleukin; LTB 4, leukotriene B 4;MIP-2, macrophage inflammatoryprotein-2; PAF, platelet-activatingfactor; PDGF, platelet-derivedgrowth factor; PMNs, polymorpho-nuclear leukocytes; PGs, prosta-glandins; TGF, transforminggrowth factor; TNF, tumor necrosisfactor.


inflammatory cells and elevated levels of mediators ofboth acute and chronic lung injury indicate that inflam-mation plays an important role in the pathogenesis ofBPD. Complex interactions between cytokines, their re-ceptors and inhibitors, and other mediators of inflamma-tion involved in the regulation of this process appear topredispose to lung tissue destruction and fibrosis. At pre-sent, one cannot speculate about the predictive value ofinflammatory cytokine measurements in infants who aredestined to develop BPD because of limited data and thecomplex network among these regulatory peptides. How-ever, studies examining cytokine activity during this pe-riod of development are increasing and will advance ourunderstanding of their role(s) in the development ofBPD.

ANTIINFLAMMATORY TREATMENT

Corticosteroids are highly effective pharmacologicagents in controlling inflammation. Because corticoste-roids are lipid-soluble molecules, they diffuse easilyacross cell membranes and bind to a specific GR in thecytoplasm.78,79This complex is then transported into thenucleus where it binds to a specific GRE containedwithin the promoter region of the target gene, resultingeither in upregulation or downregulation of gene tran-scription. In addition, there is now abundant evidencethat the GR can also function in a DNA-independentmanner to inhibit gene expression through a transrepres-sion mechanism involving protein–protein interactionsbetween the GR and the intracytoplasmic transcriptionfactors, AP-1 and NF-kB.79 These transcription factorsare known to be involved in the positive regulation of anumber of genes that play a central role in inflammation.GR transrepression mechanisms involving the transcrip-tion factors appear to be due to the formation of inactiveGR/AP-1 and GR/NF-kB complexes. Most recently, ithas been demonstrated that corticosteroids can block the

NF-kB pathway by inducing protein synthesis of its cy-toplasmic inhibitor, IkBa.80

The exact mechanism by which corticosteroids sup-press inflammation is not completely understood. Corti-costeroids inhibit transcription of a number of proinflam-matory cytokines, including TNF-a, IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, and GM-CSF.81 These effects may bemediated by interaction of GR with a negative GRE,resulting in a direct reduction in gene transcription. In-creased breakdown of mRNA has also been demon-strated for some cytokines (e.g., IL-1, IL-3, and GM-CSF).81 Corticosteroids may not only block the synthesisof cytokines, but may also block their effects in severalways.81 They may downregulate certain cytokine recep-tors (e.g., IL-2R), inhibit the activation of transcriptionfactors, such as AP-1 and NF-kB, and, furthermore, theycan directly counteract the cellular action of cytokines.Corticosteroids may also increase the synthesis of lipo-cortin-1 (a protein that has an inhibitory effect on phos-pholipase A2), and therefore inhibit the production oflipid mediators such as leukotrienes, PGs, and PAF.82

Previous controlled trials of systemic administration ofcorticosteroids at the time of diagnosis of BPD havedemonstrated improvements in respiratory status, earlierweaning from ventilatory therapy, and decreases in in-flammatory mediators in lung aspirates of infants withBPD.83–87 Systemic use of dexamethasone has becomethe treatment of choice because of its potent antiinflam-matory activity. In 1989, Cummings et al.85 conducted arandomized, double-blind, placebo-controlled study inpremature infants with BPD, using systemic dexameth-asone for 3 days (0.5 mg/kg/day), then tapering over 15or 39 days. They observed improvements in ventilatoryparameters, fewer days on mechanical ventilation, andbetter neurodevelopmental outcome at 6 and 15 monthsof age in the dexamethasone treatment group comparedto placebo. A multicenter, placebo-controlled, random-ized, and blinded study of 285 infants with BPD by theCollaborative Dexamethasone Trial Group86 demon-strated shortened duration of ventilatory support afteradministration of systemic dexamethasone (0.6 mg/kg/day IV or PO) for 1 week, followed by an optional 9-daytapering course. Groneck et al.87 examined the effects ofearly and late treatment of dexamethasone (starting witha 0.5 mg/kg/day dose and tapering over a 28-day period)on chemotaxis and inflammatory mediators in tracheo-bronchial aspirates of preterm infants at risk for BPD.They reported that most infants with RDS had elevatedchemotactic activity, neutrophils, IL-1b, LTB4, anda1-proteinase inhibitor levels in tracheobronchial fluids. Thepulmonary inflammatory response was significantly re-duced by early dexamethasone therapy (postnatal day 10).

Systemic use of corticosteroids has been associatedwith a number of adverse effects. Common transient sideeffects include hypertension, hyperglycemia, and poor

TABLE 3—Tracheal Lavage Fluid Cytokines in InfantsWith BPD*

Cytokine

Postnatal days Controlgroup(s) Ref.0–2 3–7 8–14 15–21 >22

TNF-a NS NS NS ↑ ↑ 1,2 67IL-1b NS ↑ 1 69

NS NS ↑ 1,2 74IL-6 ↑ ↑ ↑ NS NS 1,2 67

NS ↑ 1 73NS NS ↑ 1,2 74

IL-8 NS ↑ ↑ 1 65NS NS ↑ 1,2 66↑ NS 1 73

TGF-b NS ↑ ↑ NS NS 1,2 76

*NS, not significant;↑, high; 1, preterm infants with RDS; 2, infantswith nonpulmonary disease.

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weight gain. More serious side effects include adrenalsuppression, infection, myocardial hypertrophy, gastro-intestinal bleeding, and gastric perforation.88,89 In addi-tion, different studies have noted striking increases inplasma concentration of most amino acids and a decreasein urinary hydroxyproline (an amino acid found exclu-sively in collagen) in infants treated with systemic dexa-methasone for BPD.90–93These findings suggest that sys-temic dexamethasone alters protein composition eitherby suppressing protein/collagen synthesis or increasedproteolysis. However, long-term detrimental effects ofdexamethasone treatment related to impairment of pro-tein or collagen synthesis on lung growth and develop-ment in human infants are still unknown. On the otherhand, most animal studies have demonstrated that neo-natal dexamethasone treatment is associated with accel-eration of postnatal alveolar wall thinning, impairment ofpostnatal alveoli formation and alveolar surface area,suppression of cell replication (e.g., fibroblast) and di-minished conversion of type II to type I pneumo-cytes.94–97 Additionally, emphysematous changes havebeen observed as a long-term sequela in some animalstudies.97

There are a few trials of varied design using systemiccorticosteroids as a preventive therapy in infants at highrisk for BPD. Yeh et al.98 administered systemic dexa-methasone (using 1 mg/kg/day taper schedule) to prema-ture infants with severe RDS for 12 days beginning onday 1. They observed improvement in pulmonary com-pliance and earlier weaning from mechanical ventilation.BPD developed significantly less frequently in the treat-ment group. Most recently, a similar trial from the samecenter of prophylactic dexamethasone (0.5 mg/kg/day ta-pering schedule for 12 days) beginning at postnatal day 1in a group of premature infants with RDS demonstratedsignificantly higher extubation rates at day 14 of age,reduction in the incidence of BPD at 28 days, and nooxygen requirement at 36 weeks of postconceptional agein the treated group compared to the placebo group.99

Saunders et al.100 used two doses of systemic dexameth-asone (0.5 mg/kg/dose) on day 1 in low birth weightpreterm infants with RDS. The dexamethasone group re-quired fewer days of mechanical ventilation and hadshorter hospitalization, but survival without BPD was notstatistically different between treated and control groups.A recent multicenter study by Shinwell et al.101 using 3days of early postnatal systemic dexamethasone (0.5 mg/kg/day) failed to demonstrate a decrease in the incidenceof BPD. By contrast, Brozanski et al.102 observed a sig-nificant decrease in the incidence of BPD after a 3-daycourse of pulse dexamethasone (0.5 mg/kg/day) begin-ning on postnatal day 7 in ventilator-dependent infants.More recently, there have been a few trials focused onthe use of low-dose dexamethasone as an alternative tohigh-dose schedules. Durand et al.103 conducted a pro-

spective randomized study in premature infants thatfailed weaning from the ventilator at 7 or more days ofage, and compared high-dose (0.5 mg/kg/day for 3 days,0.25 mg/kg/day for 3 additional days, and 0.1 mg/kg/dayfor another day) and low-dose (0.2 mg/kg/day for 3 daysand 0.1 mg/kg/day for 4 days) systemic dexamethasonetreatment. They found that low-dose dexamethasone wasas beneficial as the high-dose regimen in shortening theduration of mechanical ventilation and that it could mini-mize the systemic side effects of corticosteroids. A pre-liminary prospective randomized trial documented asimilar time to extubation with half the dose of dexa-methasone (0.25 mg/kg/day initially, and then taperingover a 21-day period) in premature infants on mechanicalventilation with no complications as compared to histori-cal controls.104

In the last two decades, a number of controlled studieshave been conducted to assess the role of systemic cor-ticosteroids in BPD, with dexamethasone being the drugof choice, usually at a dose of 0.5 mg/kg/day. These trialshave consistently demonstrated a decrease in the numberof ventilator days and improvement in respiratory statusin the dexamethasone group. Because of the potentialside effects associated with dexamethasone, there are stillquestions regarding the best route of delivery and dose inBPD patients. Recently, alternative trials of using low-dose dexamethasone (0.2–0.25 mg/kg/day) in prematureinfants with BPD suggested similar beneficial effects ashigh-dose regimens (0.5 mg/kg/day). This may result ina reduction in adverse effects due to dexamethasone.Based on the experience of previous studies, the authorsrecommend use of a short, tapering schedule of dexa-methasone therapy at around 2 weeks of postnatal age forpremature infants on mechanical ventilation. There areongoing trials of dexamethasone administration to con-trol early lung inflammation in premature infants at highrisk for BPD. The results of these trials may potentiallychange treatment strategy in the near future.

There are increasing numbers of trials in the literature(mostly abstracts) of nebulized or inhaled corticosteroidsin infants with BPD as an alternative mode of treat-ment.105–117LaForce et al.111demonstrated improvementin lung function (dynamic compliance and airway resis-tance) by using nebulized beclomethasone dipropionatebeginning at postnatal day 14 for 28 days in ventilator-dependent premature infants. In a recently publishedstudy, Arnon et al.113 noted significant improvement inmean peak inspiratory pressure, ventilatory efficiency in-dex (VEI) and alveolar-arterial (A-a) oxygen differencein infants who were still ventilated at 14 days of agefollowing administration of inhaled budesonide for 7days.

By contrast, Kovacs et al.116 used a combination regi-men consisting of systemic dexamethasone (3 days) fol-lowed by nebulized budesonide (18 days), beginning at


postnatal day 7 in ventilator-dependent premature infantsat risk for BPD. While they observed less ventilatorysupport and oxygen requirements, and better pulmonarycompliance during the therapy period, these differenceswere not maintained over the ensuing weeks, and thedevelopment of BPD was not statistically different be-tween groups. In a randomized double-blind study, Den-jean et al.117 failed to demonstrate a beneficial effect ofcombined inhaled beclomethasone dipropionate and sal-butamol for 4 weeks in infants at risk for BPD beginningat the postnatal age of 10 days. The inhaled route oftherapy may offer advantages (with fewer side effects) inthe prevention and treatment of BPD (see reference 105for details). However, at present there is limited dataabout the absorption and distribution of inhaled cortico-steroids in distal airways and lung tissue (sites mostlikely involved in BPD).

There have been trials of antenatal maternal systemiccorticosteroid and TRH administration before pretermdeliveries. Combined use of these agents appears to actsynergistically on lung maturation and surfactant produc-tion.118,119Recent studies suggest that antenatal admin-istration of these medications reduces the severity ofBPD in low birth weight premature infants.120–122 Bycontrast, a multicenter trial by the Australian StudyGroup (ACTOBAT)123 failed to demonstrate a beneficialeffect on the incidence of RDS, oxygen requirement andneed for ventilation at 28 days of age with antenataladministration of TRH and corticosteroid treatment. Alsonoted was an increase in adverse effects in the motherswho received TRH (nausea, vomiting, lightheadedness,and rise in blood pressure). Unfortunately, significantmethodological differences have called into question theresults of these studies, such as different neonatal out-comes in subgroups, significantly greater need for as-sisted ventilation in the antenatal TRH and corticosteroidgroup compared to placebo group in ACTOBATstudy.124 Thus, the degree of benefit from antenatal useof TRH and corticosteroid in combination is unclear, andit is currently not recommended for routine use in theprevention of BPD.

Cromolyn sodium is safely used in children and adultsas an antiinflammatory medication in asthma. Whilethere are questions regarding its antiinflammatorymechanism, it seems to inhibit the release of inflamma-tory mediators from mast cells.125 The administration ofaerosolized cromolyn sodium beginning on the first dayof life in intubated premature infants with RDS failed toresult in a decrease in the incidence or severity ofBPD.126 However, limited trials have noted a reductionin inflammatory cells and mediators in aspirated trachealfluids, improvement in pulmonary function and earlierweaning from mechanical ventilation.127,128

Exogenous surfactant replacement therapy has signifi-cantly improved the survival and decreased the incidence

of RDS in premature infants. However, despite the use ofexogenous surfactant in very low birth weight prematureinfants, the incidence of BPD has not significantlychanged.129 Exogenous surfactant may suppress inflam-mation in different cell types. It has been shown in vitroto decrease lymphocyte proliferation in response to Band T cell mitogens and decrease the secretion of cyto-kines (TNF, IL-1, IL-6) by lipopolysaccharide-stimulated alveolar macrophages.130,131

A recent study has demonstrated significant superox-ide dismutase (SOD) and catalase activity in natural lungsurfactant, but not in exogenous surfactant prepara-tions.132This information may open a debate in regard tothe best treatment strategy in premature infants at highrisk for BPD (who have also been shown to have de-creased levels of antioxidant enzymes38). Indeed, sys-temic or intratracheal prophylactic administration of an-tioxidant enzymes (SOD, catalase) has been demon-strated to be effective in preventing or reducing lunginjury after exposure to high oxygen concentrations inneonatal animals.133,134 In a pilot study, Rosenfeld etal.135 observed a decrease in the severity of chronic lungdisease in a group of preterm infants receiving subcuta-neously administrated bovine SOD with no side effects.Recent animal and adult human studies further supportthe use of recombinant human superoxide dismutase (rh-SOD) by demonstrating a reduction in inflammatorycells and mediators after lung injury.136–138

There are limited studies examining the efficacy of thecombined use of exogenous surfactant and antioxidantenzymes. Using a rat model, Davis et al.139examined theeffect of exogenous surfactant on the pharmacokineticsof intratracheally administrated rh-SOD. They observedthat rh-SOD clearance or metabolism was delayed com-pared to the placebo group for at least 24 to 48 hours inexogenous surfactant treated animals, but combined usedid not alter the activity of either agent. Also, Walther etal.140 demonstrated that intratracheal surfactant lipo-somes, encapsulating SOD and catalase, resulted in asignificant elevation in the antioxidant activity in thelungs of preterm rabbits. Recently, Rosenfeld et al.141

conducted a pilot, placebo controlled study of singledoses of intratracheal by administered rh-SOD on sur-factant-treated low birth weight premature infants withRDS using two different doses. They noted that a singledose of rh-SOD significantly delayed pulmonary clear-ance or metabolism compared to the placebo group intracheal aspirate fluids, and reduced the inflammatorymediators in lung effluents without causing any discern-ible adverse effects. Unfortunately, the incidence of BPDin the rh-SOD treatment and placebo groups was notstatistically different. While the sample size of this studygroup was small and the results were discouraging, fur-ther studies with larger numbers of patients or multiple

300 Ozdemir et al.

dosing regimens are needed in order to clarify the ben-eficial effect of this new preventive therapy.

Many premature infants are deficient in vitamin A,which is important in epithelial cell growth, regenerationof damaged airway epithelium, and lung development.4

However, vitamin A supplementation in premature in-fants has not resulted in a significant reduction in theseverity of BPD.142,143 Unfortunately, the antioxidantproperties of vitamin E do not appear to change the out-come of BPD either.144,145While trials with these vita-mins are discouraging, supplementation of other traceelements (i.e., zinc and selenium) seem essential for lungdevelopment.

Ureaplasma urealyticumhas been implicated in thepathogenesis of BPD.146 The organism is commonly re-ported in vaginal flora during pregnancy and may infectpremature infants either transplacentally in utero or atdelivery, causing a chronic subclinical postnatal pneu-monia.59,147Only anectodal reports of erythromycin useappear in the literature.148Large clinical trials are neededto evaluate the role (if any) of early erythromycin treat-ment.

Recent experimental animal studies of the use of hu-man IL-8 antibody, recombinant IL-1RA or specific PAF

antagonist in lung inflammation and injury have beenencouraging. In a rat model of inflammatory lung injury,human anti-IL-8 antibody was demonstrated to be effec-tive in preventing lung injury by blocking E-selectin-dependent neutrophil recruitment.149 After ventilator-induced lung injury in rabbits, significantly lower con-centrations of albumin and elastase, and lower neutrophilcounts were observed following recombinant IL-1 an-tagonist treatment.150 In another animal study, the spe-cific PAF antagonist, BN 50739, prevented pulmonaryedema and thromboxane B2 production in IL-2-inducedlung injury in rats.151 Figure 3 illustrates a summary ofgeneral therapeutic approaches relating to the possiblefactors in the development of BPD.

FUTURE ASPECTS

Chronic lung inflammation caused by a combinationof oxygen toxicity barotrauma secondary to mechanicalventilation and inflicted on the premature infant’s imma-ture lung over a period of time seems to be the mostreasonable explanation for the development of BPD.Early, potent antiinflammatory therapy may interrupt the

Fig. 3. Summary of different therapeutic approaches in the management of bronchopulmonary dysplasia. SOD, superoxide dis-mutase; IL-1Ra, interleukin-1 receptor antagonist; anti-IL-8, anti-interleukin-8; PAF, platelet-activating factor; SLPI, secretory leu-kocyte proteinase inhibitor.


cytokine cascade and reduce the inflammatory responsein the lungs of infants who are at high risk for BPD,thereby reducing its incidence. Another alternative maybe concomitant prophylactic administration of exog-enous surfactant and rh-SOD to high-risk premature in-fants (despite the lack of efficacy in a recent pilot study).Combined use of these medications may also have anti-inflammatory effects, and decrease injury from oxygenand mechanical ventilator therapy. Finally, it may also beplausible to consider not single, but early combinationtherapy with potent antiinflammatory agents and rh-SODin high risk premature infants.

Different management strategies for the treatment ofBPD is an essential area for research. Early prophylacticuse of antiinflammatory (systemic dexamethasone, in-haled or nebulized corticosteroids) and antioxidant en-zyme therapies in infants at high risk for BPD are understudy, and will hopefully clarify the role of these newalternative approaches in the near future.

ADDENDUM

Since the submission of this manuscript, recently pub-lished articles have also suggested a possible role of GM-CSF and MIP-1a in the development of BPD.152,153

ACKNOWLEDGMENTS

We thank Dr. E. Lombardi for his helpful assistance inthe preparation of figures, Drs. E.D. Rider and J.W.Bloom for their comments and criticism, and K. Thomp-kins and I. Gomez for secretarial assistance during thepreparation of this manuscript.

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Markers and mediators of inflammation in neonatal lung disease