Chlamydia pneumoniae and Chronic Skin Wounds: A FocusedReview

Lloyd E. King Jr,* Charles W. Stratton,² and William M. Mitchell²Departments of *Medicine (Dermatology) and ²Pathology, and *Nashville Veterans Administration Medical Centers, Nashville, Tennessee, U.S.A.

The genus, Chlamydophilia, as obligate intracellularpathogens, induce chronic scarring in humans.Chlamydia pneumoniae, a common cause of pneumo-nia, infects endothelial cells and circulating macro-phages. Evidence that C. pneumoniae is anopportunistic pathogen in chronic skin ulcers and

other in¯ammatory skin conditions analogous to itsrole in atherosclerosis is reviewed. Key words:Chlamydia/chronic skin ulcers/diabetes mellitus/vasculopa-thy. Journal of Investigative Dermatology SymposiumProceedings 6:233±237, 2001

Chronic skin ulcers have common characteristics thatinclude intermittent or persistent in¯ammation,infection, and the failure to heal (Lazarus et al,1994; Mostow, 1994; Phillips, 1994; Margolis, 1996,1999a; Eaglstein et al, 1997). A nonhealing wound

may be due to genetic, environmental, or idiopathic factors, or animbalance of bacteria (Robson, 1997). Bacterial colonization ofstage 2±4 skin ulcers is assumed to be present irrespective ofetiology, so infection is a potential source of in¯ammation in allulcers such as pressure sores. How and if such in¯ammation sustainsthe most common types of chronic skin ulcers is controversial andneeds further clari®cation. The possible role(s) of Chlamydiapneumoniae in initiating or sustaining chronic skin conditions,including skin ulcers, has not been extensively investigated. Abramset al (1999) claimed that 12 of 27 patients with cutaneous T celllymphoma had a C. pneumoniae-associated protein that activatedSezary T cells. Vannucci et al (2000) detected C. pneumoniaeserologically in a diabetic patient with pyoderma gangrenosum-likelesions that responded dramatically to antibiotics directed againstChlamydia. Sams et al (2001) identi®ed by serologic, immunohis-tochemical, and culture methods C. pneumoniae in a patient withpyoderma gangrenosum that responded to prolonged antichlamy-dial antibiotic therapy with decreases in anti-C. pneumoniaeantibody titers. Serologic evidence of C. pneumoniae was retro-spectively detected by PCR and anti-C. pneumoniae IgG and IgMmethods in 13 of 20 patients with a clinical diagnosis of pyodermagangrenosum (King et al, unpublished observations). King et al(2001) also cultured C. pneumoniae from foot ulcer specimensfrom four of nine diabetic patients. We therefore propose thatC. pneumoniae is present in chronic skin conditions both as an``innocent bystander'' and as an opportunistic pathogen capable ofmaintaining in¯ammation.

CHLAMYDIA PNEUMONIAE ± ``INNOCENTBYSTANDER'', INDICATOR OF PRIOR INFECTION,

OR PERSISTENT INTRACELLULAR PATHOGEN?

Numerous studies have detected the presence of C. pneumoniae inendovascular structures as well as peripheral blood mononuclearcells (Maass et al, 1998; Boman et al, 1998; Blasi et al, 1999). Thepresence of viable C. pneumoniae in peripheral blood mononuclearcells suggests that C. pneumoniae may accompany these white bloodcells to in¯amed tissue sites and cause a secondary infection in thein¯amed tissue; however, these studies do not in and of themselvesprove a direct pathogenic role for C. pneumoniae in atherosclerosis,stroke, or other vasculopathies. For example, the prevalence rate ofC. pneumoniae in cardiovascular atheroma differing human popu-lations varies from very low (0%; Weiss et al, 1996; Lindholt et al,1998) to very high (>90%; Jackson et al, 1997) in selectedpopulations. Recent approaches focus on ®nding reliable prog-nostic tests such as immunoreactive proteins and lipopolysaccharide(LPS) or PCR detectable DNA and RNA from C. pneumoniae thatwould be indicative of active infection that is pathogeneticallyhighly relevant (Blasi et al, 1999; Shor and Phillips, 1999; Russell,1999). The data variability is due to the multiple dif®culties inculturing C. pneumoniae (Maass and Dalhoff, 1995; Maass andHarig, 1995) and technical problems with immunohistochemicalmethods (Taylor-Robinson and Thomas, 1998). Maximizing thedetection of reaction product by antigen retrieval strategies usingimmunochemical methods may be necessary in some anti-C. pneumoniae antibody staining procedures in formalin-®xedtissues as ®xation can denature antigenic epitopes of C. pneumoniae.

MICROBIOLOGIC FEATURES OF CHLAMYDIA

The genus, Chlamydophilia, Chlamydiaceae is rapidly expanding andnow includes nine species, ®ve of which were recently been addedin 1999 (see below). The various Chlamydia species are readilydistinguished by analysis of signature sequences in the 16S and 23Sribosomal genes (Everett et al, 1999; Kalman et al, 1999). Twospecies of Chlamydia are pathogenic in humans (C. pneumoniae, C.trachomatis) and seven are primarily pathogenic in other vertebrates(four species of C. psittacci, C. pecorum, C. muradarum sp. nov, and C.suis sp. nov) (Everett et al, 1999). Chlamydia trachomatis inducesconjunctivitis, keratitis and is the most common preventable causeof blindness worldwide (Ward, 1995). These organisms also cause

1087-0024/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

233

Manuscript received February 14, 2001; revised May 18, 2001; acceptedfor publication June 8, 2001.

Reprint requests to: Dr. Lloyd E. King Jr, Division of Dermatology,3900 The Vanderbilt Clinic, Nashville, TN 37232. Email: lloyd.king@mc-mail.vanderbilt.edu

Abbreviation: CP, Chlamydia pneumoniae.

pelvic in¯ammatory disease, endometrosis, vaginitis, urethritis, andinfertility. Chlamydia pneumoniae is a common pathogen in acutehuman upper and lower respiratory infections worldwide (Ward,1995).

As a genus the Chlamydia are obligate intracellular microorgan-isms that cause chronic and relapsing diseases in humans and otheranimals. Chlamydia have a unique biphasic life cycle withfunctionally and morphologically distinct dimorphic forms. Theextracellular form, the elementary body, is infectious butmetabolically inactive. After endocytosis, elementary bodies differ-entiate into the metabolic active reticulate body that replicates bybinary ®ssion. Chlamydiae have been thought to be intracellularATP scavengers as they contain nucleotide transport proteins andlack many enzymes of the electron transport chain necessary for denovo ATP biosynthesis (Stratton and Mitchell, 1997); however,recent data indicate that enough glucose-catabolizing enzymes arepresent in Chlamydia that have the functional capacity to producesome of their own ATP and reducing power (Tjaden et al, 1999;Iliffe-Lee and McClarty, 1999). The pathogenic signi®cance of thisrecent ®nding is unclear as Chlamydiae are known to stimulateglucose transport into the host cell to compensate for the extraenergy load on the infected cell (Ojicius et al, 1998). Moreover,cells infected with C. pneumoniae have depleted energy and thusmay be dysfunctional (Shemer-Arni and Lieberman, 1995).

PATHOBIOLOGY OF CHLAMYDIAL INFECTIONS

Chlamydia pneumoniae is a common cause of pneumonia in humans(Grayston et al, 1986) and is associated with in¯ammatory arthritisand atherosclerosis (see below). The antibody response to C.pneumoniae increases with age as measured in otherwise unselectedhuman populations (Grayston, 1992). Since its discovery just over adecade ago, C. pneumoniae has been associated with a number ofchronic diseases including some presumed to be autoimmune, suchas multiple sclerosis (Sriram et al, 1998, 1999; Stratton et al, 2000)and Reiter's disease (Stratton, 1998; Gerard et al, 1999). Chlamydialinfections including those caused by C. pneumoniae are remarkablebecause of their persistence in tissues. This feature has been beststudied in humans with trachoma that has periodic exacerbationsand remissions with progressive conjunctival in¯ammation andcorneal scarring (Beatty et al, 1994). Chlamydia trachomatis isdetected in the quiescent phase of trachoma even in the absence ofhistologic and immunologic features of in¯ammation. Themechanism(s) of chlamydial-induced tissue injury is unclear butlikely is relatively unique compared with gram(+) and gram(±)bacteria. Its lipopolysaccharide, lipid A, has unique structuralfeatures that induce only minimal endotoxin effects (Kosma, 1999;Rund et al, 1999). Chlamydia induce in¯ammation and clottingabnormalities, but no known chlamydial toxins have beenpositively identi®ed likely to be responsible for their tissueinjury-inducing effects. Read et al (2000) reported a potentialtype III secreted toxin with homology to the Shiga toxin producedby E. coli 0157:H7. Chlamydia have type III secretion mechanismsthat enable it to secrete and inject pathogenicity proteins into thecytosol of eukaryotic host cells, although the identity of theproteins secreted into the cytosol are unknown (Hackstadt et al,1997; Braavoil and Hsia, 1998; Hueck, 1998). Whether the in vivoactivation of the immune system by Chlamydia in vivo is due to heatshock proteins (Kol et al, 1998; LaVerda et al, 1999), type IIIsecreted proteins (see above, Braavoil and Hsia, 1998), or other Tcell activation mechanisms (Igieseme, 1996) is unknown.

CHLAMYDIA PNEUMONIAE AND INFLAMMATION

Signal transduction pathways are activated in endothelial cellsfollowing infection with C. pneumoniae (Krull et al, 1999). Infectionof human endothelial cells with C. pneumoniae stimulatestransendothelial migration of neutrophils and monocytes (Moazedet al, 1998; Molestina et al, 1999). Chlamydia pneumoniae infection ofvascular smooth muscle and endothelial cells activates NF-kB andinduces tissue factor and plasminogen activator inhibitor 1 (PAI-1)

expression (Dechend et al, 1999). Other pathogenic mechanismsthat may be involved include chlamydial heat shock proteins (Kolet al, 1998; LaVerda et al, 1999) and Toll receptors that are involvedin the innate immune responses (Muzio et al, 2000). Endothelialcytotoxicity immune reactions mediated by serum antibodies toheat shock proteins of Escherichia coli and C. pneumoniae and otherouter membrane proteins, were proposed as a possible link betweeninfection and atherosclerosis (Christiansen et al, 1999; Mayr et al,1999). Chlamydial heat shock protein 60, and potentially outermembrane proteins (Christiansen et al 1999), are involved in thelocalization to and in human atheromas and regulates macrophagetumor necrosis factor-a and matrix metalloproteinase expression(Kol et al, 1998; Christiansen et al, 1999). A speci®c C. pneumoniaelipopolysaccharide, lipid A, induces macrophage foam cell forma-tion (Kalayoglu and Byrne, 1998). A plausible explanation for thechronic and relapsing nature of C. pneumoniae infection despiteantibiotic therapy was lacking until recently. Chlamydia pneumoniaeantigens remain accessible to immunocytes for at least 4 wk and arecapable of sustaining in¯ammation when no viable C. pneumoniaeare present (Wyrick et al, 1999).

CHLAMYDIA PNEUMONIAE, VASCULARINFLAMMATION, AND ATHEROSCLEROSIS

Atherosclerosis is now classi®ed as an in¯ammatory disease (Ross,1999) so potentially it could be induced or modulated by aninfectious agent. The association of C. pneumoniae with vasculardisease was ®rst demonstrated serologically in a Finnish population(Saikku et al, 1988). Chlamydia pneumoniae was ®rst identi®ed as arespiratory pathogen by Grayston et al (1986), It was con®rmed as acommon cause of respiratory disease that was commonly detectedin asymptomatic healthy adults (Hyman et al, 1995). This organismwas ®rst directly associated with atherosclerotic lesions in SouthAfrican patients (for a review see Shor and Phillips, 1999). Recentevidence indicates that C. pneumoniae and potentially othermicrobes are associated with atherosclerosis in cardiovascular andcerebrovascular diseases (for reviews see Danesh et al, 1997; Chenget al, 1998; Epstein et al, 1999; Chiu, 1999; Grayston and Campbell,1999; Leinonen and Saikku, 1999; Bartels et al, 2000). Convincingdata that other infectious agents such as Mycoplasma pneumoniae andHelicobacter pylori may be involved are not available (Blasi et al,1996). In contrast, culture, serologic, immunocytochemical,polymerase chain reaction (PCR), in situ hybridization, andtransmission electron microscopy data combined with epidemio-logic data show that C. pneumoniae is detected in a statisticallysigni®cant number of samples from human arterial tissues includingperipheral vessels (Shor and Phillips, 1999). Chlamydia pneumoniaeinfection is detectable in patients with cerebrovascular accidents(stroke) (Cook et al, 1998) as well as Alzheimer's disease (Balin et al,1998) and diabetic nephropathy (Kanuchi et al, 2000). Therefore,C. pneumoniae is likely to be widely prevalent in the vasculature.

PATHOGENETIC SIGNIFICANCE OFATHEROSCLEROSIS AND C. PNEUMONIAE IN

DIABETICS

It has been claimed that the endovascular presence of C. pneumoniaeDNA is a generalized phenomenon in atherosclerotic vasculardisease (Maass et al, 1998). These data are intriguing as athero-sclerosis is the underlying cause of chronic limb ischemia and oftenis the cause of chronic ulcers in many patients. The progression ofatherosclerosis is accelerated by the coexistence of hypertension,lipoprotein abnormalities, tobacco addiction, and diabetes mellitus(Kempczinski and Bernhard, 1995). Although atherosclerosis is notqualitatively different in diabetics, it appears at an earlier age andprogresses more rapidly. Peripheral vascular disease (PVD) indiabetics most commonly affects the tibial, popliteal, and profundafemoris arteries rather than the aorta and iliac arteries (Kempczinskiand Bernhard, 1995). Atherosclerosis is generally assumed to be thecause of chronic ischemia of the lower extremities, includingdiabetic foot ulcers (Chait and Bierman, 1994). There are very few

234 KING ET AL JID SYMPOSIUM PROCEEDINGS

collateral vessels in the lower leg so even nonsegmental occlusion ofthe distal and proximal tibial arteries may result in gangrene orsevere ischemia requiring arterial reconstruction (Kempczinski andBernhard, 1995).

IS C. PNEUMONIAE LIKELY TO BE THE ONLYPATHOGEN INVOLVED IN ATHEROSCLEROSIS?

Multiple infections of atherosclerotic plaques may be present(Epstein et al, 1999; Chiu, 1999; Zhu et al, 2000). Serum levels ofhigh-sensitivity C reactive protein (hs-CRP), an acute phasereactant, correlated well with the total pathogen burden or numberof chronic endogenous pathogens and coronary artery risk (Zhuet al, 2000). In a much larger study, the levels of hs-CRP were asaccurate a predictor of coronary artery disease in a long-term studyof the coronary risk factors as cholesterol and lipid pro®le in apopulation of nurses (Ricker et al, 2000).

DETECTING C. PNEUMONIAE IN CHRONIC LEG ANDDIABETIC FOOT ULCERS

The previous inability to detect these organisms may be due to thedif®culty of recovering viable C. pneumoniae organisms that requirespecial methods for their isolation and propagation (Stephens,1992). Studies on C. pneumoniae were aided by the discovery of ahuman lung cancer cell line, HL, that allowed isolation andpropagation of the TWAR strain of C. pneumoniae (Kuo andGrayston, 1990). Chlamydia pneumoniae persistently infects andreplicates in epithelium, endothelium, smooth muscle cells,macrophages (Sriram et al, 2000), and neural tissue in vivo andin vitro (Balin et al, 1998; Sriram et al, 1998, 1999; Stratton et al,2000). The ability to isolate and propagate these organisms in HLcells (Kuo and Grayston, 1990; Pruckler et al, 1999; Sriram et al,2000) requires additional centrifugation steps and a 7 d culturetime, resulting in a 500±5000-fold increase in the number ofdetectable inclusion-forming units. As punch or excisional biopsiesare relatively contraindicated in patients with evolving or resolvingdiabetic foot ulcers, routine curettage samples of diabetic foot ulcersare especially helpful as a standard source for culture.

SOURCE OF C. PNEUMONIAE IN CHRONIC SKINULCERS

If C. pneumoniae is to be considered as a source of infection forchronic skin ulcers, then the origin of the C. pneumoniae infection isgermane. Chlamydia pneumoniae is common in the environment(``innocent bystander''), dif®cult to eradicate, and may be spread bycontact with seemingly healthy humans (Hyman et al, 1995).Alternatively and, we believe, most likely, C. pneumoniae in chronicskin ulcers may be initiated by parasitized mononuclear cells(Airenne et al, 1999). In a mouse model, C. pneumoniae infectivitywas demonstrated to be mediated by parasitized monocytes(Moazed et al, 1998). A possibility that has not been previouslyconsidered is that extracellular elementary bodies may benoncovalently bound or attached to circulating RBC via inter-action with membrane molecules such as heparin.

IS A SPECIFIC SKIN TYPE CELL THE SOURCE OFC. PNEUMONIAE IN CHRONIC SKIN ULCERS?

Chlamydia pneumoniae was detected in human keratinocytes,endothelial cells, and histiocytes that contained intracellular inclu-sions stained with anti-MOMP (outer membrane protein) and anti-LPS (Abrams et al, 1999). These authors con®rmed the presence ofC. pneumoniae DNA and RNA in skin by PCR and RT-PCR andproductively infected keratinocytes in vitro with C. pneumoniae(Abrams et al, 1999). Recently, we detected C. pneumoniaeserologically and by PCR in chronic cutaneous ulcers in a diabeticwho responded dramatically to appropriate antibiotic therapy(Vanucci et al, 2000). We cultured C. pneumoniae from chronic

skin wounds in diabetic and nondiabetic patients such as those withpyoderma gangrenosum (King et al, 2001; Sams et al, 2001).

DOES C. PNEUMONIAE INFECTION PREDIPOSEPATIENTS TO DEVELOP CHRONIC SKIN ULCERS?

This is the fundamental question that underlies the debate overwhether C. pneumoniae is an ``innocent bystander'' or an oppor-tunistic pathogen (see reviews cited above). As demonstrated by thecon¯icting data and opinions over the role of C. pneumoniae inatherosclerosis and coronary artery disease, it is important to ®rstdocument that an active infection by C. pneumoniae is unequivoc-ally present at the site of tissue damage. Similarly, the ability todocument the evolving pathologic responses or nonresponses toC. pneumoniae is critical to interpretation of epidemiologic data.Studies on the vasculopathy of coronary arteries, the aorta, and theabdominal aorta are severely limited by their accessibility forde®nitive biopsies and long-term follow-up. Epstein Barr virus,Cytomegalovirus, Herpes simplex type I and II, and potentiallyother members of the herpes virus family as well as Hepatitis Avirus were proposed as opportunistic agents or copathogens forchronic vasculopathies (Zhu et al, 2000). Antibody titers, PCRtiters, and culture results do not always directly correlate especiallywith patients receiving high doses of immunosuppresants (Sams andKing, unpublished data). No published data are currently availableto document the presence of chronic C. pneumoniae skin infectionsin diabetics or other causes of chronic leg ulcers so more study isrequired to evaluate this possibility.

CHLAMYDIA PNEUMONIAE, SKIN INFECTIONS,OR ULCERATIONS AND DIABETES

No direct data are available to con®rm that there is an increase inC. pneumoniae skin infections in the chronic leg ulcer population;however, there are known associations between DM andatherosclerosis as well as between C. pneumoniae and atherosclerosis(Muhlestein, 1998; Shor and Phillips, 1999). Indirect evidencesuggests that increased blood glucose due to DM may help sustainpersistent C. pneumoniae infections to compensate for the extraenergy load on infected cells (Ojicius et al, 1998). Chlamydialinfections increase glucose consumption, lactate production,glutamate synthesis, glycogen accumulation, and an associatedincrease expression of the glucose transporter, GLUT-1, in vitro(Ojcius et al, 1998). Conversely, diabetics are assumed to be verysusceptible to infections so that it is not unreasonable to assume thatC. pneumoniae are among the unusual and/or opportunisticpathogens that might be detected in some diabetics with chroniculcers. There may be an association, moreover, between chronicC. pneumoniae infections and diabetic nephropathy based upon anti-C. pneumoniae serum IgG antibody titers in an ELISA (Kanuchi et al,2000).

CHLAMYDIA PNEUMONIAE, SERUM LIPIDS, DM, ANDATHEROSCLEROSIS

Abnormal cholesterol and lipoproteins are major risk factorsassociated with atherosclerosis in diabetic as well as and nondiabeticpatients. Atherosclerotic plaques contain foam cells and evidence ofaltered lipid composition (Ross, 1999). Animal models (Moazed etal, 1997; Saikku et al, 1998; Muhlestein et al, 1998; Fong et al, 1999)show that C. pneumoniae can induce atheromas and mimicin¯ammatory processes present in human atherosclerosis. ElevatedC. pneumoniae antibody titers were associated with altered serumlipid pro®le in humans (Laurila et al, 1998). In vitro cellularoxidation of low-density lipoproteins (LDL) is induced byC. pneumoniae (Kalayoglu et al, 1999). The atherogenic effects ofChlamydia are claimed to be dependent on serum cholesterol andspeci®c to C. pneumoniae (Hu et al, 1999).

VOL. 6, NO. 3 DECEMBER 2001 CHLAMYDIA PNEUMONIAE AND CHRONIC SKIN WOUNDS 235

THERAPY

Published data to con®rm the ef®cacy of chronic antibiotic therapyin the treatment of coronary artery disease to eliminate chronicinfection are inconclusive. Chlamydia pneumoniae use monocytes asa transport for systemic dissemination and enter a persistent statenot covered by an otherwise effective antichlamydial treatment, soprevention of vascular infection by such treatment may beproblematic (Gieffers et al, 2001). Which antibiotic(s) to use intreating atherosclerosis is a moot point as multiple infectious agentsmay be involved and induce an unacceptably high pathogen burden(Chiu, 1999; Zhu et al, 2000). Chlamydia pneumoniae infected cellsalso may be more resistant to apoptosis than cells infected withother pathogens (Geng et al, 2000). Although chronic antibiotictherapy did alter the clinical course of multiple skin ulcers withserologically documented C. pneumoniae infection with (Vannuciet al, 2000) and without diabetes (Sams et al, 2001), it is not clearhow many other patients would respond. Standard treatment ofchronic venous ulcers or diabetic foot ulcers may not heal themwithin 12 wk (Margolis et al, 1999b), so long-term, cost-effectivetherapy may be required. The ef®cacy and cost-effectiveness ofspeci®c regimens for chronic skin ulcers will likely be a productiveresearch area.

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