MA[Awl MEDJoURNAt;1 5(2)68-Zt JUNE. 2OO3
Hiddenrisksfor pneumoniain Malawi DGFullertonr,SBGordon, 1. Departmentof RespiratoryMedicineHopeHospital, stott Lane,salford,M6 gHD,u.K. 2. Malawi-Liverpool-Wellcome ClinicalResearch Programme,Universityof Malawi,Blantyre,Malawi. Domestic smoke exposure and early HIV infection are criti_ cal but unseen risk factors for pneumonia. This paper reviews how recent research in Malawi and elsewhere con-
Introduction Lower respiratory tract infections account for a major burden of prematuredeathand disability worldwide(,).The greatestburden of diseaseis felt in the developingworld where the incidenceof acute lower respiratory tract infections (ALRI) is 12 fold higher than in developedcountries(2).Risk factors for ALRI include age (0-11 months),gender(male),lack of breasrfeeding,HIV infec_ tion, malnutrition (both macro and micro-nutrients)and envi_ ronmental factors such as crowding and indoor air pollutiol{:'1. In Malawi, endemic HIV infection and the common use of smoky fuel for both cooking and lighting are the most common risk factors for respiratorytract infections in adults. This review will focus on how important pulmonary defencemechanismsare compromisedby theserisk factors.
tributes to an understanding of the possible mechanisms underlying these risks.
Figure I PanelA
Normallung defence The lung is constantly challenged by inhaled particles and microbial pathogens. The first line of defence consists of mucus-coveredciliated epithelium and the cough reflex(5).The next line of defenceconsistsof innate responsesthat are non_ specifichost defenceresponsesto foreign particlesoccuringpre_ dominantly at the epithelial surface. Mediators of the innate responseinclude soluble factors and host cells(or). Finally, humansmount acquiredresponsesto invadingpathogens.These are specifichumoral (antibody)and opsono-phagocytic respons_ es regulatedby the cellular immune system. The entire immune responsemust rapidly remove inflammatory stimuli becausethe resprratorytract is a fragile tissue with a delicate structurethat is desisnedfor gas exchangeand excessiveinflammation impairs this function(8).
Airway clearance,domestic smoke and tobacco smokingin Malawi Each day approximately 7000 litres of air are drawn through the nose u'here large particles are deposited. particulate material larger than 0.5_m in diameter is depositedin the lining fluid (mucus)of the tracheaand bronchi{o).Clearanceof this debris, paniclesand secretionsis by mucociliary action. The lower air_ r\-avs are ciliated and lined by epithelium that contains goblet cells and submucosalglands. These glands produce enough mucus to cover underlying cilia which constantly sweep the mucus towardsthe throat where it is swallowed(10). In Malawi, the majority of homes use a mixture of fuels for cooking including wood and charcoal, as well as a variety of smoky means of lighting including paraffin tin lamps and candles("). The extent of this smoke exposureca! be clearly demonstratedby examining fluid washedfrom the lungs of patientsat bronchoscopyas shown in Figure 1. The effect of this smoke on the ciliary func_ tion and mucus production of exposedMalawians has not yet been assessed.
Figure 1 This figure shows bottles of freshly obtained bronchoalveolar lavage taken from 2 different subjects on the same day, and alveolar macrophages obtained from the same samples stained with the fluorescent nucleic acid stain DAPI and viewed under standardtransmitted light with simultaneous laser illumination. The left hand sample is dark in color due to carbon particles within the alveolar macrophages that can be seenoutlining the cytoplasm under transmitted light (middle panel). The right hand sample is cloudy in color and the macrophage preparation shows cell nuclei staining blue together with blue-staining coagulase-negativestaphylococci which were added to the preparation. Supematantfluid after removal of the cell pellets from both samples was clear.
Mucociliary clearance is substantially impaired by cigarette smoking due to the increasedviscosity and volume of mucus produced by smokers as well as the damage to cilia causedby increasedlevelsof proteasesin the mucus(12). Cigarettesmoking is the most important risk factor for pneumonia among immunocompetent adults in the USA('r). The health significance Malawi Medical Journal
in Mw H i d d e nr i s k sf o r p n e u m o n i a
hfl
- -.:reltesrnokingin Malawi is not currently known but is an .' :1rnt area for future researchas tobacco is a major local , ,.-.r.rop and both commercialand home-rolledcigarettesmok- i. common. Clinical diagnosesof chronic bronchitis sec:,J.ln to cigarettesmoking are still rare in QECH, Blantyre. ,- )ughcan be a voluntaryaction or a reflex respiratoryresponse inhaled particulate matter, irritants, high or low temperatures .,rd humidity(s).Most commonly cough is a symptom of infec.ion. but in developednations it is associatedwith cigarette .moking. In Malawi, chronic cough is associatedwith tuberculosis"r'. The incidence of cough as a symptom was common arnongMalawianswith HIV and pneumococcaldisease,but the durationof cough was not predictiveof outcome among hospital admissions(").
69
are priniitirr- receptrlrsc-rnhort cells that respondto well-cons e r l e d s t r u c t u r e . t r i m i c r t ' r o r g a n i s n ls. u c h a s b a c t e r i a l lipopolrsacharide.lnd '-:rrbohldrlite\ or iorms of bacterial DNA. The Toll-like rec€ptor,llftr gn ihe surlaceof alveolar macrophages(TLR-I r h.r. r.'centlr L'een :hou n to mediate i n n a t e r e s p o n s e s t o - \ 1 '1, 1 , i r i ; . ;i si 1 1 r r 1i t l ' t ' r t t t l t t s i s a n d t''to'. pneunrortittt' in hunl.tnalr artllr nlacrophages Streptococcus The inflammatoryresponseof alr ltrl.rr lllicrr)lhlge: to an intracalledpn-unl(rl\:in.hasbeen cellularproductof S. ptteurnoriirre shown to be dependenton TLR-I. Lnpubli.hed data from BlantyrehaveshownthatHIV inlectedadult. hlr r' alteredbaseline expressionof TLR-2 and TLR--I but the .ignit'icanceof thesechangesin relation to pulmonarl det'enceagein.t S.ltrteuis still underinrestigatton. moniaeor M.tuberculosls
lnnateimmunefactorsand lung defencein Malawi Acquiredimmunityin the lung A rangeof solubleinnateimmune mediatorsmeetsparticlesthat Immunoglobulins are found at all levels of the respiratorl' lmmunoglobulin in the respiratorytract ag-elutinates passthrough the mucociliaryblanket. Someof thesefactorsare tract(21'3r). complementand actsas an opsoninfor phagoactivates induced by spebacteria, at low levels and others are producedconstantly resident alveolarmacrophagesor neutrophilsmi-sratby g in 1921 cytosis first discovered by Flemtn cific activation. Lysozyme, , parenchyma in responseto inflammationr'. IgA is polyinto the ing is a bactericidalprotein capableof lysing the carbohydrate trect mers that comprise the external membrane of bacteria('t"'t). the predominantimmunoglobulin of the upper respirator-1' epitheliRespiratory parenchyma. in the predominates protease IgG inhibitor and Lactoferrin and secretory leucocyte (SLPD are airway defenceproteinsproducedby serouscells as um facilitatesthe secretionof secretorylgAffr. IgG is thoughtto n'ell as neutrophils. Lactoferrin is able to kill and agglutinate be produced locally by pulmonary plasma cells that have maturedin regionallymph nodesand migratedback to the tissue bacteria,which it recognizeson the basisof carbohl'dratemotifl '''. where antigen was first presented(ta).In acute inflammation, ihe as well as stimulatingsuperoxide productionb1 neutrophils Both lactoferin and lysozyme are produced in nuch -qreater epitheliumbecomesincreasinglypermeableand so plasmaproquantitiesin patientswho havechronic bronchitis'". The a- and teins.includingalbuminand immunoglobulin,leak in to the resB- defensins show broad anti-microbialactivity a-eainstGram- piraton tract'j. Immunoglobulinfacilitatesphagocytosisby receptorson macrophagesand neunegative and Gram-positivebacteria, mycobacteriafun-si and bindin,sto imrnuno-elobulin They act by inducing permeabilisationand are t r o p h i l . . some virusestzor. recunentrespiratoryinfections up regulated in the lung in response to the inflantmator\ In patient:nith IgG detrciencr'. cytokine,interleukin-1(IL-l;'''r. The collectins are a famrlr oi lre a nrajor problem' . In studiesof HIV patientsfrom the that IgG levelsin lung fluid proteinsthat bind to carbohydrateson the surfaceof pathogen:. US-\. some earll uork su-g-qested rThis triggersthe alternatecomplementcascade.Collectinsalso might be low in patients i.rith HIV . Several studies have have direct effectson the activationof immune cells rncludin-e therefore measuredthe levels of pulmonary immunoglobulins macrophagesand lymphocytes.Ke) rrembersof this family both in serum and the lung fluid of Malawian adults. In include - surfactant proteins A and D (SP-A and SP-D) and Malawian adults with HIV infection, increasedlevels of IgG, IgM and IgA were found in plasma and lung fluid. The lung mannan-bindinglectin (MBL) " " . Severalof theseinnate factors have recently been examinedin fluid levels were not significantly alteredby correction (using lung fluid from Malawian volunteersto determinetheir relation albumin levels) for the effect of plasma leakage. In addition, with carbonexposureand HIV infection. No significantdiffer- pneumococcal polysaccharidespecific IgG was particularly ence was found in the level of lactoferrin,lysozyme or SLPI in increasedin the lung following recent pneumococcaldisease a comparisonbetween HIV negativeadults and HIV positive both in HIV infected and control patientsbut the increasewas adultswith or without a recenthistory of pneumococcaldisease significantly greater in HIV infected adults. This increasein (manuscriptsubmitted). In HIV negativeadults,however,there total and specificIgG was thoughtto be due to the increasedprowas an increasedlevel oflactoferrin in subjectswith high carbon duction of IL-6 in HIV infected adults- this cytokine drives B loads in alveolarmacrophages.This suggestedthat exposureto cell antibodyproduction(r8).There is evidencefrom other parts smoke stimulateslactoferrin production in Malawians despite of the world that genetic variations in the immunoglobulin the low incidenceof chronicbronchitis. Thereis scopefor much receptorstructurecan increasethe risk of pneumoniain certain This work will begin shortly in Blantyre. more work on innate pulmonary defencefactors in Malawian populationson). adults. Complement proteins are important componentsof the innate and acquiredimmune defenceand are found within lung secrePatientswith complementdeficiencies(eg sickle cell tions{2ar25). patients) experiencerecurrent infectionswith capsulateorganStreptococcus isms including Haemophilus inJTtrettzae'!"'and pneumoniaett''. There havebeen no studiesto dateof complement levels and function in the lungs of Malawian adults. Host cells are alsocapableof mountinga cellular innateimmune r e s p o n s e . S e v e r a lr e c e p t o r sc o n t r i b u t e t o t h i s r e s p o n s e (eg scavengereceptor,mannosereceptor)but the recently disThese coveredtoll-like receptors are of particularinterest('?8). Malawi Medical Journal
C e l l u l a rc o m p o n e n t so f a c q u i r e di m m u n i t yi n t h e lung Macrophages,dendritic cells and lymphocytes act together in the lung to orchestratethe rapid uptake and removal of foreign particlesfrom the lung. The proportionof alveolarmacrophages and the extent of activation of these populationsin adult lung fluid is the subjectof current study in our laboratory. As previo u s l y d e s c r i b e d ,H I V i n f e c t e d a d u l t s h a v e i n c r e a s e d lymphocytesand B cells in lung fluid, with a vastly increased percentageof CDS expressingT lymphocytesia''4lr. Alveolar macrophages are the predominant phagocyte of the
-'
lil]l/td@n rbfs for pneumonia in Mw
- ,:.,]'::p"r"-i;1ndare tbund at a densityof approximatelyone hours,peaking at 12-18hours. -llr. -\lveolar macrophagesingest small numbers :':: '-',r,.,,r.is :::11;1.:. Ltacteriaor apoptotic cells and releaseoxidative Cytokines, chemokines and the acute inflammatory responsr *=::,r and proteaseson to this material in phagolysosomes(25). in the lung S r::: :acteria le.g. Legionella spp and Mycobacteriaspp) can In the face of overwhelming infection, macrophagesand lvrn :-,> rare pha_eolvsosomal processing,either by resistanceto the phocytes generatepro-inflammatory cytokines to recruit polr::r5:r,-isome contentsor by escapeinto the cytosol(43). Following morphonuclear and monocytes. Cytokines are peptides pro.t_gesiionrn the phagosome,macrophagespresent antigen to duced by metabolically active cells of the immune system1i mphocl'teseitherlocally or in the regionallymph node(*).The They have the ability to activate other cells and include proteins precise mechanism by which antigen is transported to the such as interleukins, interferons, tumour necrosis factor, and regional lymph nodes is poorly understoodin humans,but is chemokines. Their effects include chemotaxis, degranulation. likeli to involve dendritic cells and other pulmonary protein production, cell division and activation, cytoskeletal macrophages (interstitial macrophages). Overwhelmed reaffangement and immunomodulation. Cytokines usually acr macrophages undergo apoptosis and are removed by the over short distances and bind fo target cell receptors. Recent mucociliaryescalatorand coughreflex(*s). Apoptosisis a prefer- work in Malawi has shown that alveolar macrophagesfrom Hf\' able responsecomparedto necrosisas it reducesthe releaseof infected adults produced decreasedIl-lbeta in responseto inflammatory mediators that might otherwise damagethe respi- S.pneumoniaecompared to HIV negative adults. In addition. ratory epithelium(46). HIV infected adults were found to have increasedRANTES (a HfV infects and affects alveolar macrophagestaT). This leads to beta-chemokine) in lung fluid - these pro-inflammaton altered function ofreceptor expression,activation, cytokine pro- responsesare probably appropriate responsesto the challenge duction, accessorycell function, phagocytosisand apoptosisof posed by the viral infection itselfl"). Carbon loading of alveolar the macrophagesthemselves. Phagocytosisand killing of macrophages has not yet been associated with alterations in Cryptococcus neoJbrmans(8)and Pneumocystis cariniiqe) was cytokine production in Malawi but this is under investigation. seento be impaired in alveolar macrophagesfrom HIV infected Acute smoke exposure increased circulating neutrophil counts subjects, but no impairment of Staphylococcus aureu,r(so) or by a bone malrow effect mediated by increasedIL-8 production S.pneumoniae(51) was observed. Some differences in alveolar by alveolar macrophagesin a study from Singapore(tt). macrophage function may be due to the prevailing cytokine milieu in the alveolus and so these findings may have to be lnteractionof carbonexposureand HIVin the lung repeated in the light of different lymphocyte populations and in Malawi cytokine measurementsin the lung fluid. Carbon has a pro-inflammatory effect on the lung as evidenced An important aspect of alveolar macrophage function that is by altered lactoferrin and RANTES levels. HIV is associated often overlooked is anti-inflammatory cytokine productienrs'?r. with increasedimmunoglobulinlevelsand CD8 lymphocytes. It Alveolar macrophageshave a predominantly immunosuppres- is interesting to speculatethat the two influences might interact sive influence on the alveolar surfacethat is altered in favour of and indeed there is a difference in the number of alveolar phagocytosis and inflammation in the presence of bacteria or macrophagescontaining large amounts of carbon between HIV viruses. No difference in anti-inflammatory profile was found, infected and non-infected Malawians (seeFigure 2). however, in the responseof Malawian alveolar macrophagesto Figure 2 S.pneumoniaein vitro (unpublished data). Lymphocytes. In normal individuals, lymphocytes account for I0% of cells harvestedfrom bronchoalveolarlavage(BAL). In addition, lymphoid aggregatesand follicles containing mainly BJymphocytes are found in contact with the visceral surface of the epithelial layer - this is known as bronchus-associatedlymphoid tissue (BAL1;o'r. The lymphocytes harvestedfrom BAL are approximate\y 997o T-lymphocytes. These cells distinguish self from foreign protein antigen presentedby macrophagesand B cells to cell surface antigen receptors (TCR). Different subpopulations of T-lymphocytes defined by surface markers have different functions. CD4 expressingcells are helper T cells with a predominant role in regulating antibody responsesand activating macrophages. CD8 expressing lymphocytes are cytotoxic cells and produce a cytokine profile that activatesmacrophages and NK cells to an effective anti-viral response(s').
E mean ca.bon %
b!
!
o !
L
HIV IIIV negative
HIV positive
Figure 2
Neutrophils and monocytes make up only l-27o of lung lavage The mean percentageof macrophagescontaining carbon is compared from healthy individuals but the vast majority of cells in acutely betweeh HIV infected and uninfected subjects. There is a higher percentage in non-HlV-infected subjects. Dividing subjects about the pneumonic lungtttr. A large number of neutrophils are median in to high or low carbon, a greater proportion of high carbon sequesteredby the fluid dynamics and small capillaries of the containing samples are obtained from HIV negative subjects (Pearson pulmonary circulation and so are effectively on "stand-by" near chi=4.2:p=0.039). Monocytes are immature macrophagesthat the airspace(56). circulate like lymphocytes and neutrophils and form an important immune reserve, rapidly recruited when necessary. The mechanismsto explain why HIV infected Malawians have less carbon in their macrophagescould include down-regulation Monocyte emigration follows the neutrophils, starting at 6 Malawi Medical Joumal
Hiddenrisksfor Pneumoniain Mw ' receptors or decreasedmacrophagelifetime but there is no :', r,Jenceto date to support these hypotheses.
5ummary
Erposure to smoke and HIV infection provide a significant challenge to the immune mechanisms defending healthy lung. Srudying the mechanismsby which theseeffects occur will pror ide further information about lung defence in health and diseaseand may generateinformation leading to new approachesin the preventionoI pneumonia.
receptor 2-deficient mice are highly susceptible to Streptocmcus pneumonlae meningitis becauseof reduced bacterial clering md enhmced inflarnmation' J.lnfect-Ds. l00l: I 86t61:798-806. Reiling \. Holrher C- Fehanbmh -\- Kmger S. Kirschning CJ' Goyert S et al' pathogen recognition in Cunin! edge: Toll-lile recepor ITLR rl- and TlRtmediated tuberculosis' J'Immunol' misrare io airbom int-ecdu qith \llcoba"rerim
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Hiddenrisksfor pneumoniain Malawi DGFullertonr,SBGordon, 1. Departmentof RespiratoryMedicineHopeHospital, stott Lane,salford,M6 gHD,u.K. 2. Malawi-Liverpool-Wellcome ClinicalResearch Programme,Universityof Malawi,Blantyre,Malawi. Domestic smoke exposure and early HIV infection are criti_ cal but unseen risk factors for pneumonia. This paper reviews how recent research in Malawi and elsewhere con-
Introduction Lower respiratory tract infections account for a major burden of prematuredeathand disability worldwide(,).The greatestburden of diseaseis felt in the developingworld where the incidenceof acute lower respiratory tract infections (ALRI) is 12 fold higher than in developedcountries(2).Risk factors for ALRI include age (0-11 months),gender(male),lack of breasrfeeding,HIV infec_ tion, malnutrition (both macro and micro-nutrients)and envi_ ronmental factors such as crowding and indoor air pollutiol{:'1. In Malawi, endemic HIV infection and the common use of smoky fuel for both cooking and lighting are the most common risk factors for respiratorytract infections in adults. This review will focus on how important pulmonary defencemechanismsare compromisedby theserisk factors.
tributes to an understanding of the possible mechanisms underlying these risks.
Figure I PanelA
Normallung defence The lung is constantly challenged by inhaled particles and microbial pathogens. The first line of defence consists of mucus-coveredciliated epithelium and the cough reflex(5).The next line of defenceconsistsof innate responsesthat are non_ specifichost defenceresponsesto foreign particlesoccuringpre_ dominantly at the epithelial surface. Mediators of the innate responseinclude soluble factors and host cells(or). Finally, humansmount acquiredresponsesto invadingpathogens.These are specifichumoral (antibody)and opsono-phagocytic respons_ es regulatedby the cellular immune system. The entire immune responsemust rapidly remove inflammatory stimuli becausethe resprratorytract is a fragile tissue with a delicate structurethat is desisnedfor gas exchangeand excessiveinflammation impairs this function(8).
Airway clearance,domestic smoke and tobacco smokingin Malawi Each day approximately 7000 litres of air are drawn through the nose u'here large particles are deposited. particulate material larger than 0.5_m in diameter is depositedin the lining fluid (mucus)of the tracheaand bronchi{o).Clearanceof this debris, paniclesand secretionsis by mucociliary action. The lower air_ r\-avs are ciliated and lined by epithelium that contains goblet cells and submucosalglands. These glands produce enough mucus to cover underlying cilia which constantly sweep the mucus towardsthe throat where it is swallowed(10). In Malawi, the majority of homes use a mixture of fuels for cooking including wood and charcoal, as well as a variety of smoky means of lighting including paraffin tin lamps and candles("). The extent of this smoke exposureca! be clearly demonstratedby examining fluid washedfrom the lungs of patientsat bronchoscopyas shown in Figure 1. The effect of this smoke on the ciliary func_ tion and mucus production of exposedMalawians has not yet been assessed.
Figure 1 This figure shows bottles of freshly obtained bronchoalveolar lavage taken from 2 different subjects on the same day, and alveolar macrophages obtained from the same samples stained with the fluorescent nucleic acid stain DAPI and viewed under standardtransmitted light with simultaneous laser illumination. The left hand sample is dark in color due to carbon particles within the alveolar macrophages that can be seenoutlining the cytoplasm under transmitted light (middle panel). The right hand sample is cloudy in color and the macrophage preparation shows cell nuclei staining blue together with blue-staining coagulase-negativestaphylococci which were added to the preparation. Supematantfluid after removal of the cell pellets from both samples was clear.
Mucociliary clearance is substantially impaired by cigarette smoking due to the increasedviscosity and volume of mucus produced by smokers as well as the damage to cilia causedby increasedlevelsof proteasesin the mucus(12). Cigarettesmoking is the most important risk factor for pneumonia among immunocompetent adults in the USA('r). The health significance Malawi Medical Journal
in Mw H i d d e nr i s k sf o r p n e u m o n i a
hfl
- -.:reltesrnokingin Malawi is not currently known but is an .' :1rnt area for future researchas tobacco is a major local , ,.-.r.rop and both commercialand home-rolledcigarettesmok- i. common. Clinical diagnosesof chronic bronchitis sec:,J.ln to cigarettesmoking are still rare in QECH, Blantyre. ,- )ughcan be a voluntaryaction or a reflex respiratoryresponse inhaled particulate matter, irritants, high or low temperatures .,rd humidity(s).Most commonly cough is a symptom of infec.ion. but in developednations it is associatedwith cigarette .moking. In Malawi, chronic cough is associatedwith tuberculosis"r'. The incidence of cough as a symptom was common arnongMalawianswith HIV and pneumococcaldisease,but the durationof cough was not predictiveof outcome among hospital admissions(").
69
are priniitirr- receptrlrsc-rnhort cells that respondto well-cons e r l e d s t r u c t u r e . t r i m i c r t ' r o r g a n i s n ls. u c h a s b a c t e r i a l lipopolrsacharide.lnd '-:rrbohldrlite\ or iorms of bacterial DNA. The Toll-like rec€ptor,llftr gn ihe surlaceof alveolar macrophages(TLR-I r h.r. r.'centlr L'een :hou n to mediate i n n a t e r e s p o n s e s t o - \ 1 '1, 1 , i r i ; . ;i si 1 1 r r 1i t l ' t ' r t t t l t t s i s a n d t''to'. pneunrortittt' in hunl.tnalr artllr nlacrophages Streptococcus The inflammatoryresponseof alr ltrl.rr lllicrr)lhlge: to an intracalledpn-unl(rl\:in.hasbeen cellularproductof S. ptteurnoriirre shown to be dependenton TLR-I. Lnpubli.hed data from BlantyrehaveshownthatHIV inlectedadult. hlr r' alteredbaseline expressionof TLR-2 and TLR--I but the .ignit'icanceof thesechangesin relation to pulmonarl det'enceagein.t S.ltrteuis still underinrestigatton. moniaeor M.tuberculosls
lnnateimmunefactorsand lung defencein Malawi Acquiredimmunityin the lung A rangeof solubleinnateimmune mediatorsmeetsparticlesthat Immunoglobulins are found at all levels of the respiratorl' lmmunoglobulin in the respiratorytract ag-elutinates passthrough the mucociliaryblanket. Someof thesefactorsare tract(21'3r). complementand actsas an opsoninfor phagoactivates induced by spebacteria, at low levels and others are producedconstantly resident alveolarmacrophagesor neutrophilsmi-sratby g in 1921 cytosis first discovered by Flemtn cific activation. Lysozyme, , parenchyma in responseto inflammationr'. IgA is polyinto the ing is a bactericidalprotein capableof lysing the carbohydrate trect mers that comprise the external membrane of bacteria('t"'t). the predominantimmunoglobulin of the upper respirator-1' epitheliRespiratory parenchyma. in the predominates protease IgG inhibitor and Lactoferrin and secretory leucocyte (SLPD are airway defenceproteinsproducedby serouscells as um facilitatesthe secretionof secretorylgAffr. IgG is thoughtto n'ell as neutrophils. Lactoferrin is able to kill and agglutinate be produced locally by pulmonary plasma cells that have maturedin regionallymph nodesand migratedback to the tissue bacteria,which it recognizeson the basisof carbohl'dratemotifl '''. where antigen was first presented(ta).In acute inflammation, ihe as well as stimulatingsuperoxide productionb1 neutrophils Both lactoferin and lysozyme are produced in nuch -qreater epitheliumbecomesincreasinglypermeableand so plasmaproquantitiesin patientswho havechronic bronchitis'". The a- and teins.includingalbuminand immunoglobulin,leak in to the resB- defensins show broad anti-microbialactivity a-eainstGram- piraton tract'j. Immunoglobulinfacilitatesphagocytosisby receptorson macrophagesand neunegative and Gram-positivebacteria, mycobacteriafun-si and bindin,sto imrnuno-elobulin They act by inducing permeabilisationand are t r o p h i l . . some virusestzor. recunentrespiratoryinfections up regulated in the lung in response to the inflantmator\ In patient:nith IgG detrciencr'. cytokine,interleukin-1(IL-l;'''r. The collectins are a famrlr oi lre a nrajor problem' . In studiesof HIV patientsfrom the that IgG levelsin lung fluid proteinsthat bind to carbohydrateson the surfaceof pathogen:. US-\. some earll uork su-g-qested rThis triggersthe alternatecomplementcascade.Collectinsalso might be low in patients i.rith HIV . Several studies have have direct effectson the activationof immune cells rncludin-e therefore measuredthe levels of pulmonary immunoglobulins macrophagesand lymphocytes.Ke) rrembersof this family both in serum and the lung fluid of Malawian adults. In include - surfactant proteins A and D (SP-A and SP-D) and Malawian adults with HIV infection, increasedlevels of IgG, IgM and IgA were found in plasma and lung fluid. The lung mannan-bindinglectin (MBL) " " . Severalof theseinnate factors have recently been examinedin fluid levels were not significantly alteredby correction (using lung fluid from Malawian volunteersto determinetheir relation albumin levels) for the effect of plasma leakage. In addition, with carbonexposureand HIV infection. No significantdiffer- pneumococcal polysaccharidespecific IgG was particularly ence was found in the level of lactoferrin,lysozyme or SLPI in increasedin the lung following recent pneumococcaldisease a comparisonbetween HIV negativeadults and HIV positive both in HIV infected and control patientsbut the increasewas adultswith or without a recenthistory of pneumococcaldisease significantly greater in HIV infected adults. This increasein (manuscriptsubmitted). In HIV negativeadults,however,there total and specificIgG was thoughtto be due to the increasedprowas an increasedlevel oflactoferrin in subjectswith high carbon duction of IL-6 in HIV infected adults- this cytokine drives B loads in alveolarmacrophages.This suggestedthat exposureto cell antibodyproduction(r8).There is evidencefrom other parts smoke stimulateslactoferrin production in Malawians despite of the world that genetic variations in the immunoglobulin the low incidenceof chronicbronchitis. Thereis scopefor much receptorstructurecan increasethe risk of pneumoniain certain This work will begin shortly in Blantyre. more work on innate pulmonary defencefactors in Malawian populationson). adults. Complement proteins are important componentsof the innate and acquiredimmune defenceand are found within lung secrePatientswith complementdeficiencies(eg sickle cell tions{2ar25). patients) experiencerecurrent infectionswith capsulateorganStreptococcus isms including Haemophilus inJTtrettzae'!"'and pneumoniaett''. There havebeen no studiesto dateof complement levels and function in the lungs of Malawian adults. Host cells are alsocapableof mountinga cellular innateimmune r e s p o n s e . S e v e r a lr e c e p t o r sc o n t r i b u t e t o t h i s r e s p o n s e (eg scavengereceptor,mannosereceptor)but the recently disThese coveredtoll-like receptors are of particularinterest('?8). Malawi Medical Journal
C e l l u l a rc o m p o n e n t so f a c q u i r e di m m u n i t yi n t h e lung Macrophages,dendritic cells and lymphocytes act together in the lung to orchestratethe rapid uptake and removal of foreign particlesfrom the lung. The proportionof alveolarmacrophages and the extent of activation of these populationsin adult lung fluid is the subjectof current study in our laboratory. As previo u s l y d e s c r i b e d ,H I V i n f e c t e d a d u l t s h a v e i n c r e a s e d lymphocytesand B cells in lung fluid, with a vastly increased percentageof CDS expressingT lymphocytesia''4lr. Alveolar macrophages are the predominant phagocyte of the
-'
lil]l/td@n rbfs for pneumonia in Mw
- ,:.,]'::p"r"-i;1ndare tbund at a densityof approximatelyone hours,peaking at 12-18hours. -llr. -\lveolar macrophagesingest small numbers :':: '-',r,.,,r.is :::11;1.:. Ltacteriaor apoptotic cells and releaseoxidative Cytokines, chemokines and the acute inflammatory responsr *=::,r and proteaseson to this material in phagolysosomes(25). in the lung S r::: :acteria le.g. Legionella spp and Mycobacteriaspp) can In the face of overwhelming infection, macrophagesand lvrn :-,> rare pha_eolvsosomal processing,either by resistanceto the phocytes generatepro-inflammatory cytokines to recruit polr::r5:r,-isome contentsor by escapeinto the cytosol(43). Following morphonuclear and monocytes. Cytokines are peptides pro.t_gesiionrn the phagosome,macrophagespresent antigen to duced by metabolically active cells of the immune system1i mphocl'teseitherlocally or in the regionallymph node(*).The They have the ability to activate other cells and include proteins precise mechanism by which antigen is transported to the such as interleukins, interferons, tumour necrosis factor, and regional lymph nodes is poorly understoodin humans,but is chemokines. Their effects include chemotaxis, degranulation. likeli to involve dendritic cells and other pulmonary protein production, cell division and activation, cytoskeletal macrophages (interstitial macrophages). Overwhelmed reaffangement and immunomodulation. Cytokines usually acr macrophages undergo apoptosis and are removed by the over short distances and bind fo target cell receptors. Recent mucociliaryescalatorand coughreflex(*s). Apoptosisis a prefer- work in Malawi has shown that alveolar macrophagesfrom Hf\' able responsecomparedto necrosisas it reducesthe releaseof infected adults produced decreasedIl-lbeta in responseto inflammatory mediators that might otherwise damagethe respi- S.pneumoniaecompared to HIV negative adults. In addition. ratory epithelium(46). HIV infected adults were found to have increasedRANTES (a HfV infects and affects alveolar macrophagestaT). This leads to beta-chemokine) in lung fluid - these pro-inflammaton altered function ofreceptor expression,activation, cytokine pro- responsesare probably appropriate responsesto the challenge duction, accessorycell function, phagocytosisand apoptosisof posed by the viral infection itselfl"). Carbon loading of alveolar the macrophagesthemselves. Phagocytosisand killing of macrophages has not yet been associated with alterations in Cryptococcus neoJbrmans(8)and Pneumocystis cariniiqe) was cytokine production in Malawi but this is under investigation. seento be impaired in alveolar macrophagesfrom HIV infected Acute smoke exposure increased circulating neutrophil counts subjects, but no impairment of Staphylococcus aureu,r(so) or by a bone malrow effect mediated by increasedIL-8 production S.pneumoniae(51) was observed. Some differences in alveolar by alveolar macrophagesin a study from Singapore(tt). macrophage function may be due to the prevailing cytokine milieu in the alveolus and so these findings may have to be lnteractionof carbonexposureand HIVin the lung repeated in the light of different lymphocyte populations and in Malawi cytokine measurementsin the lung fluid. Carbon has a pro-inflammatory effect on the lung as evidenced An important aspect of alveolar macrophage function that is by altered lactoferrin and RANTES levels. HIV is associated often overlooked is anti-inflammatory cytokine productienrs'?r. with increasedimmunoglobulinlevelsand CD8 lymphocytes. It Alveolar macrophageshave a predominantly immunosuppres- is interesting to speculatethat the two influences might interact sive influence on the alveolar surfacethat is altered in favour of and indeed there is a difference in the number of alveolar phagocytosis and inflammation in the presence of bacteria or macrophagescontaining large amounts of carbon between HIV viruses. No difference in anti-inflammatory profile was found, infected and non-infected Malawians (seeFigure 2). however, in the responseof Malawian alveolar macrophagesto Figure 2 S.pneumoniaein vitro (unpublished data). Lymphocytes. In normal individuals, lymphocytes account for I0% of cells harvestedfrom bronchoalveolarlavage(BAL). In addition, lymphoid aggregatesand follicles containing mainly BJymphocytes are found in contact with the visceral surface of the epithelial layer - this is known as bronchus-associatedlymphoid tissue (BAL1;o'r. The lymphocytes harvestedfrom BAL are approximate\y 997o T-lymphocytes. These cells distinguish self from foreign protein antigen presentedby macrophagesand B cells to cell surface antigen receptors (TCR). Different subpopulations of T-lymphocytes defined by surface markers have different functions. CD4 expressingcells are helper T cells with a predominant role in regulating antibody responsesand activating macrophages. CD8 expressing lymphocytes are cytotoxic cells and produce a cytokine profile that activatesmacrophages and NK cells to an effective anti-viral response(s').
E mean ca.bon %
b!
!
o !
L
HIV IIIV negative
HIV positive
Figure 2
Neutrophils and monocytes make up only l-27o of lung lavage The mean percentageof macrophagescontaining carbon is compared from healthy individuals but the vast majority of cells in acutely betweeh HIV infected and uninfected subjects. There is a higher percentage in non-HlV-infected subjects. Dividing subjects about the pneumonic lungtttr. A large number of neutrophils are median in to high or low carbon, a greater proportion of high carbon sequesteredby the fluid dynamics and small capillaries of the containing samples are obtained from HIV negative subjects (Pearson pulmonary circulation and so are effectively on "stand-by" near chi=4.2:p=0.039). Monocytes are immature macrophagesthat the airspace(56). circulate like lymphocytes and neutrophils and form an important immune reserve, rapidly recruited when necessary. The mechanismsto explain why HIV infected Malawians have less carbon in their macrophagescould include down-regulation Monocyte emigration follows the neutrophils, starting at 6 Malawi Medical Joumal
Hiddenrisksfor Pneumoniain Mw ' receptors or decreasedmacrophagelifetime but there is no :', r,Jenceto date to support these hypotheses.
5ummary
Erposure to smoke and HIV infection provide a significant challenge to the immune mechanisms defending healthy lung. Srudying the mechanismsby which theseeffects occur will pror ide further information about lung defence in health and diseaseand may generateinformation leading to new approachesin the preventionoI pneumonia.
receptor 2-deficient mice are highly susceptible to Streptocmcus pneumonlae meningitis becauseof reduced bacterial clering md enhmced inflarnmation' J.lnfect-Ds. l00l: I 86t61:798-806. Reiling \. Holrher C- Fehanbmh -\- Kmger S. Kirschning CJ' Goyert S et al' pathogen recognition in Cunin! edge: Toll-lile recepor ITLR rl- and TlRtmediated tuberculosis' J'Immunol' misrare io airbom int-ecdu qith \llcoba"rerim
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