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Experimental onchocercal keratitis
Experimental Onchocercal Keratitis E. Pearlman may resolve spontaneously. Prolaged, chronic in&mmation, however, can rffult in scarring (sclerosing) keratitis which is the primary cause of blindness due to onchocerciasis’s.
Invasion of the eye by Onci~oc~rcn uolo~rl~rs microfilariae (Mfl can result in either anterior segment disease, ie. inflammation of the cornea (keratitis), or posterior segment disease, ie. inflammation of the retina, choroid or weal tract. Although some reference will be made to posterior segment disease, this review will focus on the immunopathology of keratitis, which accounts for most cases of blindness associated with cmclwcerciasi.+. The pathophysiological mechanisms underlying the clinical features of keratitis (ie. cornea1 opacification and neovascularizationl can be considered in terms of loss of the normal transparent, avascular nahxe of the cornea. Transparency of the mammalian cornea is dependent on the orderly arrangement of collaeen fibrils in the cornea1 stroma. which Dennits light-to penetrate without diffraction’. Main’tenance of the fibrillar arrangement is dependent on a critical level of hvdration in the cornea1 stroma. which is controlled’in part by a Na+/K+-ATPasedependent pump in the cornea1 endothelial cells. During acute keratitis, it is likely that release rf mediators from inflammatory cells disrupts the normal level of hydration, resulting in comeal edrwa and rearrange ment of collagen fibrils. l’enetratir>: light would then be diffrwted, raulting in the conma having an opaque appearance Growth of blood vessels into the comeal stroma may also contribute to disruption of fibrillar organization. A subepithelial cell infiltrate develops around the Mf, causing a pnnctate keratitis, which
Human onchacercal keratitis The World Health Organization estimates that 27OCCO people are blinded fmm this disease and 5CQwO are severely visually impaired, mostly due to sclemsing kemtiti3. In some villages in hyperoldemk areas of Africa, up to 10% of inhabitants have serious vision impairment as a result of onchocerciasis. Although keratitis accounts for most cases of blindness, the parasites can also induce damage to the choroid, retina and ontic new+. Eoidemiokxical and entomological s&dies have sh&n ttat k&&is is more prevelent in the savanna regions of Africa, whereas hinitis is more common in the rainforest areas of Africa”‘“. Evidence for inherent eenetic differences between strains has been shown cy the use of DNA probes that can distinmdsh between rainforest and Savanna forms of 0. mlTirlus”.‘~. Ocular pathology c-xurs when 0. uolvelas Mf enter the cornea after migrating through the pertorbital skin and conjunctiva. So long as they remain alive, Mf elicit little or no infiammatory response, and motile v.wms can be detected by routine slit-lamp examination in the ccnnc~ of infected individuals with no apprent inflammation. When the Mf die, a number of antieens are then likelv to be released which, in a host th\t has been sensit~ed by chronic exposure to the parasites, may then stimulate an inflammatory response. In support of this notion, treatment with diethvlcarbamazine rapidly kills the Mf and is assocatcd ‘with punctate keraiitis, ie. discrete areas of cornea1 opacification that resolve spontanwusly with minimal visual impairments? These lesions comprise local edema with infdtrating lymphocytes and eosincphils”. In contrast, sclerosiing keratitis OCCUTS ir. heavily infected individuals where there is prolonged invasion of the wrnea by large numbers of Mf resulting in an intense inflammatory response, scarring and severe visual impainnent’~ (Fig. 11. While it has been speculated that, in endemic areas, pu+te le+ons
Overview of animal models Although natural infection with 0. rwlorrlus has been recorded and chimpanzees and cynomolgus monkeys can be infected, these nnimals do not develop ocular d&ease’“,?“. Therefore, there is no established animal model of natural. 0. z&rrlrrs-mediated ocular oathology. To study the pathogenesis of onchocercdl’ker.7titis, invrstitqtors have either inoculated live Mf into the conjun&” or cornea of laboratory animals, or injected sohlble parasite antigens into the cornea1 stroma. This approach induces keratitis within days or weeks, as compared with years for human infection. In general, intraconjunctival injection with live Mf induced punctate keratitis, whereas direct intracorneal injection of Mf or soluble sntieens induced a more severe inflammation resembling’&lerosing keratitis. Many of these stndies are outlined in Table 1.
Investigation o. ‘nunune mechanisms underlying onchocercal keratitis (&Cc? -. ~ nctate 3 sclerosing) has been hampered by the difficulty in obtaining corneas from infected individuals. Many of the corneal samples are from end-stage disease, when the corneas are mostlv fibrotic. Studies have therefore used adjacent cohjuncti:al tissue, and have demonstrated abscesses compnsing dead Mf with eosinophils and T cells were also found to infiltrate the lymphocytes’“. conjunct& of patients, and resident cells were in II stati of activadon as measured by major histocomoabbilitv com&x (MHC) class II exoressioa”. Chan if nl. d&ted bterleukin 4 (IL-4) mRkA in conjunctivae from seven out of ,en ot these patients, but found no correlation with disease statu9, However, IL-4 and IL-5 production by peripheral blood mononuclear cells (PBMC) stimulated with parasite antigen were found to o,rrelate positively with ocular and dermal disease tD.0. Freedman, pers. commun.). These studies indicate that IL-r (which induces B-cell switch to IgE srcretion) and IL-5 (which stimulates eosinophil production) are likely to be involved in immunopathologj of orular onchocerciasis.
Primates as models Donnellv and co-workers have rcoorted a series of experirne& in which live Mf of either 0. oulwhrs or the cattle pawite 0. lirrmlis wcie inoculated into various sites ;n the eyes of cynomolgus monkeys”,‘“‘“. Animals injected intracomea!ly showed a minimal inflammatory response, even if they had been previously immunized”. The absence of corneal lesions in these singly challenged animals suggests that chronic invasion of large numbers of Mf is necessary for develooment of lesions. These results are consistent with’ human disease, but differ from sh-dies using guinea-pigs inoculated with live 0. licne’is Mf (see below). The prtmatc model rn”v be more useful or studying posterior disease, as qection of 0. liwnlis Mf into the vitreous or pusterior areas of the eye induces a pronounced inflammatory reqxmse comprising monw cyts and densely packed maws of eusinophils. Tiiese inflammatory lesions are often seen to surround Mf in the vilreous area, indicating a role for thee cells in parasite cleamnc@. Comparison of rainforest and savanna strains Duke and co-workers were among the first to use animal models to study the development of 0. ralo~d~rs
Reviews mediated keratiti+x. These investigators recovered live 0. oolvr~lrrs Mf from skin snigs of volunteers from either the savanna or the rainforest region of Cameroon. Microftlartae WlG3X-O) were then inoculated directly into the conjunctiva of mbbitsat several sites around the cornea. Although only a small percentage of the inoculated Mf invaded the cornea, about three times more savanna strain parasites than rainforest strain Mf were found to be present (O&4.5% vs O.l-2.4%). In addition, the savanna strain parasites induced a more intenae inflammation, characterized by punctate keratitis, comeal vascularization and occasional stromal keratitis. Histoloeical exam cat% of corneas showed that infiltration t? infiammatory cells, primarily eosinophi!s and lymvhocytes, was greater in rabbits in&&ted with- the savanna strein than with the minforest strain2’. Jnoculation of dead Mf directly into the cornea1 stloma YesuIted in relatively mild lesions wth no differences in clinical or Qath&Jgical reaction observed between the two stmins (kemtiiis did not develop if killed Mf were injected into the conjunctiva). The difference in virulence between these strains is therefore more likely to be clue to the invasive chamcteristics of ~~annn strain Mf than to intrinsic differences in Qrwasite rmtigens between the rtmins. This conclusion is also consistent with observations ot posterior eye disease induced after intravitreal and subretinal inlettion of live Mf into rabbits. Although thse animals develooed an inflammatorv resoonse. there was no differ&e between savanna and ramforest strains of the pamsite”~~~~. To determine the effect of pre-existing immune raponses on the dcvelopmcnt of kcratitis, rabbits were immunized intravenously with live Mf prior to intraconjunctival challenge. These animals displayed punctate kentitis one zlzy after injection compared with four days in unimmunized animals. lr addition to the accelerated appe.~nnce of &ease in immunized rabbits, the severity of keratitis was enhanced and contained a prominent neutrophil infiltmte”. In contrast to injection with live Mf, rabbits immunized intmvenously with dead Mf appeared to be tolerized to subsequent exposure to live Mf, which is a more typical outcome of intravenous injection. These animals had only minimal ocular pathology, and motile Mf were detected in the cornea up to 17 days after inoculation. Rodent models using Mf Given the difficulty in obtaining sufficient numbers of 0. WIVUIIW Mf from infected individuals, subsequent studies have used Mf recovered from horses or cattle. lntraconjuncttval inoculation of rabbits and guinea-pigs with 0. gettwusn Mf from cattle did not ip duce keratitis”. O~rl~ocwcn cervicalis Mf isolated from horses invade the comeal stroma of CSA/H micp after subcutaneous injection, but no inflammatory response was detected in these animals, even after repeated injection and treatment with diethylcarb amazine (DEW (E.R. James, per% commun.). In contrast to studies descrtbed above using 0. gtrtterosn and 0. ccrvicnlis, Mf of the caltIe parasite 0. licruk penetrate the cornea and induce tipacities resembling human punctate keratitia when inoculated intmconjunctivally into Hartley strain guineaPoronltologyTodoy.vol. I2. “0 7. 1996
pig+‘“. Histological examination of the lesions reveals foci of eosinophils and mononuclear cells together with local edema, which is similar to that reported for human onchocercte.sislJ.~. Repeated subconjunctival inoculation with live 0. limnlis Mf c.auses more rapidly aQ;2earing and severe pun&ate lesions, whereas sut.xtaneuus immumzation suppressed the development of keratitis after subconjunctival inoxul&ion. Sakla c: n1.z suggest that the immune response elicited by subcutaneoi~s immunization results in death of the Mf before they can penetrate the cornea. Although it is not known why the immune response to subconjunctival immunization would then result in eracc:bated pathology, it is possible that the Qamsites are not killed in the conjunctiva, but are able to penetrate the cornea and become the focus of an inflammatory response at that site. In contmst to observations with subconjunctivally iniccted Mf, direct injection of live Mf into the correal btroma of subcutaneously immunized guinea-pigs resulls in an intense keratitis, resembling scterosing keratitls in hilmans2”‘. These animals dwelop nea vascularizaticn and opacities covering up to half the cornea. The opacities are found to be associated with comeal edema and recruitment of eusinophils, neutrophils, lymphocytes and plasma cells. Keratitis is not induced unisss the animals either are previously sensitized or have rweived spleen cells from immunized animals prior to intracomeal inoculation, demonstrating thnt celluiar responses are required for develop ment of !.eratitiP’. Cornea1 inflammation also cormlatch with increased IgE levels both in sera and in aqueous humor, indicating that this antibody isotype is involved in mediating keratitis. The QESEmce of IgE and wainophils in this model is consistent with human disease manifestations and with production of IL-4 and IL-3 (Refs 40,411. The capacity for 0. limnlis Mf to induce keratitis in guinea-pigs, but not in cynomolgus monkeys, may be due to subtle anatomic differences in the corneas of Qrim.,teS and rodentsn. The investigators suggest that Bowman’s membrane, which underlies the comeal epithelium. is thick- in primates and humans than in rodents, and is relatively redstant to neovascularization and subsequent keratitis. Rodent models using 0. oolvwlus antigens The observations in human infection and in the animal models (described above) demonstrate that keratitis is induced after Mf havr ratered the cornea and died, releasing internal mtigens. Injection of 0. uolr~drrs antigens COvAgl directly into rhe cornea could then substitute for live Mf. Using this approach, unsensitized guinea-pigs and mice have a mild, inflammatory response with minimal stromal infiltrate, whereas animals receiving prior immunization with OvAg develop pronounced cornea, opaciticatton and neovasculartzatton26~“’ (Fig. 2). The inflammatory response is similar to that described above for live Mf. The composition of the cellular infiitrate was dependent on the adjuvant used during systemic immunization, as animals @en a single immunization of OvAg in complete Freuncls adjuvant prior to inb;lcomeal injection develoQ+d kemtttis in which neutrophils are present during tlw initial three days, and lymphocytes, plasma cells and 163
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histiocytes are present after seven dayrWq. In contrast, mice given repeated immunizations of OvAg in a squalene based adjuvant (with no mycobacterial antigens) prior to intracomeal injection develop a maximal response on Days 4-10, and the cellular infiltrate is uredominantlv eosinauhils (Fig. 3)“‘. The difference ‘m cellular in&ate b&wee” &he two models has yet to be determined. The role of CDl+ T cells and IL-4 Chakravartt and co-workers have shown that CD4’ T cells rather than CD8+ T cells are predominant in the corneas of A/J mice throughout the course of keratitis, implicating a role for these cells in the inflammatory responses’. On the basis of cytokine product:z, CD4* T cells can be grouped into T helper cell tyFe 1 CThl) and type 2 (Th2) subtypes. Thl cells produce interferon y (IFN-$, implicated in delayed-
type hypersensitivity reactions, whereas Th2 cells oroduce IL-4 and IL-5. which are reauired for mohuction of IgE and eos&mphils, respedively~~. A;described above, eosinophils are prominent in inflamed corneas of rabbits and guinea-pigs inoculated with live MPQ7.11 and IgE is present in the aqueous humor in infected guinea-pig+“. These observations suggest that induction of ThZ-cell-associated r~ponses are involved in development of kemtitis. A more direct assessment of the Th-cell response induced by 0. vnfvul~rs antigens has been determined by examining cytokine production by lymph node and spleen cells from BALB/c mice immunized subcutaneously with OvAg)‘. OvP.g-stimulated CD4+ T cells produce IL-4 and IL-5, but not IFN?, thereby demonstrating a predominant antigen-specific ThZcell response. Cytokine pmduction at the site of iiulamnation is determined by reverse tmnscription
Reviews PCR (polymerase chain reaction) using corneas removed at the peak cf the inflammatarv restw”se (ie. seven days after in&&wd injection). OvAg-injected corneas have elevated levels of CD4’ and CDEV T cells and IL-4 gene expression compared with unbjected corneas from the sane animals (Fig. 41, although uninjected corneas demonstrate CD8 expression. Consistent with in vilro cytokine production, IFN-y expression remains low, indicating that ThZ- rather than Thcell responses dominate at the site of inflammation. As IL-4 is required for development of Th2 cells, its role in 0. uoluzrlwmediated stromal keratitis has been determiwd in mice in which the iL-4 gene has been disrupted? In contrast to control C5781/6 and F2 mice, IL4-deficient mice develop significantly less keratitis than do control mice, thereby demonstrating an essential role for IL-4 in immunopathology”. Histological examination reveals that the cornea1 stromas of IL4-deficient mice are not edematous and contai” very few inflammatory cells (E. Pearlman, a:qxblished), suggesting that IL-4 may facilitate entry of inflammatory cells into the comeal stroma. Link between and keratitis I
,
Th2-cell
.
r CD4
Q
CD8
w
IFN-r
-
IL-4
a
HPRT
m
responses
.
ad on known functions of r&dent cells in the cornea, a sequence of immunwnediited events leading to keratitis has been propoSea (Box 11. Repeated or chronic exposure to OvAe induces a oredominant ThZcell ~&~nse sys&cally, with elevated serum IgE and eosinophilia, and IL4 and 1LSpmducing CD4f T cells. Injection of parasite antigen into the comeal stroma, or active penetration by live Mf, may stimulate cornea1 epithelial cells to produce inflammatory cytokinff such as IL-1 and tumor necrosis factor u (TNFn), which stimulate comeal iibmblasts (keratocytes) to release chemokima and enhance the expression of intercellular adhesion molecule-l (ICAM-1) and vascular adhesion molecule-l (VCAM-I) on vascular end&&l cells. IL-la and TNFa stimulate keratocyte pm&cticm of RANTES (regulated upon activation, normal T expressed and weted) and MCP-I (monocyte chemotactir protehW* which attract lymphuiy is and eosinophils in oilro. IL-la and TNF-a also stimulate integrin expression on human umbilical endothelial cells grmw in uitrd’, and are likely to have a similar effect on capillary venules. Upregulation of ICAM-I and VCAM-I on end&h&l cells may facilitate adherence and transmigration of wsinophils and OvAgspecific Th2 cells into the cornea1
Box 1. Predicted Sequence of Events Leading to Onchocercal Keratitis Svstemic rewonse ‘Repeated i;nmu”iratio” with parasite, 0. m,or,,r,s, anti&en (OvAg, selectively induces a ThZ-cell resplnse chronic infection in human disease). * OvAg-specific Th2 memory cells circulate through the vasculah~re.
*
(mimics
Immediate response at site of i+ctin” * Injection of parasite antigen int” the comeal stroma UTactive penetration of live Mf tigers release of inflammatory cytokines IL-I a”d TNF-u from cwneal epithelial cells. . IL-1 and TNF-a induce stromal f&v&lasts (kemtacvtcs, to ~roducc chemokines which attract Ivm~hocvm and I eosinophils. Inflammatory cytokines als” stimulale expression of molecuuln on capillary ve”“les. * MHC class LIpositive La”gerha”s ce11smigrate from the limb”.? to the cornea, stnnna. * A”giogenic factors are released. I
*
.
.
adhesion
Infiltration of inflammatory cells . OvAS-spedfic Th2 cells and eosinophils b,“d m capillary endothehal cells and migrate to the romeal stroma. * Class II paitiw La”gcrh.,“s EclLspiesent atigen to T cells. . Activated Th2 cells secrete cytukine and chemokines, leading tc further recmihnent of inflammatory cells. . IL-4 sti”ukIte5 further Thz-cell pralifemtio”. ThZ-cell-mediated inflammatory response * Eosinophils degranulate, releasing eosinophil major basic protein and o,her mediators into the strcona, matrix. . Release of i”flammat”ry mediators disrupts the co!lage” fibril arrangemm~ * Carneal edema ensues, resulting in loss of transparency. * Further release of angiogrnic factors stimulates mmeal “mvascularizati”“.
and comeal endothelial cell function.
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injury
stroma, where they may migrate to the site of by following a chemokine gradient. Further stimulation of Th2 cells at this site would require antigen presentation. As nnther fibroblasts nor cornea1 epithelial cells express MHC class II moldepends on migrstion of ecules, antigen presentation either monocytes from the circulation or, more likely, migration of Langerhans dendritic cells. Langerhans cells are potent antigen-presenting cells which reside in the conjunctive and cornea1 limbus, and which migrate to the central cornea in response to injury or infection’. Antigen presentation activates OvAgspecific Th2 cells, leedins to IL-4-mediated T-cell proliferation and productmn of IL-5 and RANTES, and rasinopbil recruitment. Degranuhtion of eosinophils would release mediators, including ma@ basic protein, eosinophil cationic protein and eosinophil peroxidase. These can be expected to disrupt stromal tibril arrangement either directly (by interacting with the collagen and proteoglycan). or indinxtly (by impeding the function of the Na’ /K*-ATl’ase-depadcnt pump in corncal endothelial cells, thereby increasing the hydration level of the cornea1 stroma). This scenario is con&tent with the observed comeal edema. In either case. verturbathrt: cf the collagen fibril arrangement will &It in loss of transparency and a clinical picture of cornea1 op.xification~ The factors underlying cornea1 neovasculatiation in onchocercal keratitis have yet to be determined. Given the variety of cell types present, it is likely that an array of angiogenic factors are involved, iwILding IL-I, IL-Z, IL-S, TNF-ol and tmnsforming growth factor p (TGF-PPH”y. In addition to production of angiogenie factors, the inflammation may have an indirect role in disrupting the balance of angiogenic and angiostatic factors that is essential for the maintenance of the avascular nature of the normal cornea. Other factors, such as autoimmune responses, may also contribute to the observed pathology.
Relation to human disease Although the predicted sequence of events is derived primarily from observations in animal mod&, there are at least three features that are common to human disease: (1) the requirement for prior sensitization (as a result of either chronic infection or prior immunization), which results in an appropriate cellulx response; (2) the presence of parasite antigens in the cornea1 stroma either after invasion of live Mf or by direct injection of parasites or parasite antigen; and (3) development of a sustained inflammatory response in the corn& Worna. A more complctc understanding of these events may suggest a rationale for immune-based intervention which could potentially limit development of 0. vulwdus-mediated ocular disease.
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Lymphatic Filariasis: What
Mice Can Tell Us R.A.
Law-ence m.&nisms used by the parasite will make it possible to tareet strateeies for immune intervention. The use of an&al mod& is an essential link in our understanding of immunity to filartasis, because it is only in these systems that we can combine knowledge of the immune rejponse with knowledge of the duration of infection, the developmental stages present, the infection dose and the site of infection. An ideal animal model of lymphatic filariasis would ha&our the full developmental cycle and dismimplay similar pathology to humans in addition icking the immunological characteristics of human infection. In the absence of a convenient laboratory model with these attributes, a surprising amount has been learnt from the study of short-term infections in mice. With the advent of genetically manipulated mice in which specific pathways of the immune Esponse have been deleted, the importance of precisely defined immune parameters can be assessed in &xctective immunity, parasite sup and host pathology. This review draws together our current knowledge of tilariasis in the mouse, and highlights areas in which further research can provide answers to the major questions in lymphatic filariasis as well as give new direction to human studies
to
Iid
and to determine the CBUSBof the patholdgical res~cmsesseen in others. Furthermore, the parasite SUP v&es at the heart of the Immune system &I extended periods of time. Identification of the immune evasion
The filarial parasites Brrrgin sp. and W. bnncrofC are transmitted by mosquito vectors. Infective stage larvae (L3) initiate infection when the mosquito feeds upon the human host. The larvae migrate to the lymphatic vessels and mature into adult male and female worms. After mating, the female worms prcduce
Invasion of the eye by Onci~oc~rcn uolo~rl~rs microfilariae (Mfl can result in either anterior segment disease, ie. inflammation of the cornea (keratitis), or posterior segment disease, ie. inflammation of the retina, choroid or weal tract. Although some reference will be made to posterior segment disease, this review will focus on the immunopathology of keratitis, which accounts for most cases of blindness associated with cmclwcerciasi.+. The pathophysiological mechanisms underlying the clinical features of keratitis (ie. cornea1 opacification and neovascularizationl can be considered in terms of loss of the normal transparent, avascular nahxe of the cornea. Transparency of the mammalian cornea is dependent on the orderly arrangement of collaeen fibrils in the cornea1 stroma. which Dennits light-to penetrate without diffraction’. Main’tenance of the fibrillar arrangement is dependent on a critical level of hvdration in the cornea1 stroma. which is controlled’in part by a Na+/K+-ATPasedependent pump in the cornea1 endothelial cells. During acute keratitis, it is likely that release rf mediators from inflammatory cells disrupts the normal level of hydration, resulting in comeal edrwa and rearrange ment of collagen fibrils. l’enetratir>: light would then be diffrwted, raulting in the conma having an opaque appearance Growth of blood vessels into the comeal stroma may also contribute to disruption of fibrillar organization. A subepithelial cell infiltrate develops around the Mf, causing a pnnctate keratitis, which
Human onchacercal keratitis The World Health Organization estimates that 27OCCO people are blinded fmm this disease and 5CQwO are severely visually impaired, mostly due to sclemsing kemtiti3. In some villages in hyperoldemk areas of Africa, up to 10% of inhabitants have serious vision impairment as a result of onchocerciasis. Although keratitis accounts for most cases of blindness, the parasites can also induce damage to the choroid, retina and ontic new+. Eoidemiokxical and entomological s&dies have sh&n ttat k&&is is more prevelent in the savanna regions of Africa, whereas hinitis is more common in the rainforest areas of Africa”‘“. Evidence for inherent eenetic differences between strains has been shown cy the use of DNA probes that can distinmdsh between rainforest and Savanna forms of 0. mlTirlus”.‘~. Ocular pathology c-xurs when 0. uolvelas Mf enter the cornea after migrating through the pertorbital skin and conjunctiva. So long as they remain alive, Mf elicit little or no infiammatory response, and motile v.wms can be detected by routine slit-lamp examination in the ccnnc~ of infected individuals with no apprent inflammation. When the Mf die, a number of antieens are then likelv to be released which, in a host th\t has been sensit~ed by chronic exposure to the parasites, may then stimulate an inflammatory response. In support of this notion, treatment with diethvlcarbamazine rapidly kills the Mf and is assocatcd ‘with punctate keraiitis, ie. discrete areas of cornea1 opacification that resolve spontanwusly with minimal visual impairments? These lesions comprise local edema with infdtrating lymphocytes and eosincphils”. In contrast, sclerosiing keratitis OCCUTS ir. heavily infected individuals where there is prolonged invasion of the wrnea by large numbers of Mf resulting in an intense inflammatory response, scarring and severe visual impainnent’~ (Fig. 11. While it has been speculated that, in endemic areas, pu+te le+ons
Overview of animal models Although natural infection with 0. rwlorrlus has been recorded and chimpanzees and cynomolgus monkeys can be infected, these nnimals do not develop ocular d&ease’“,?“. Therefore, there is no established animal model of natural. 0. z&rrlrrs-mediated ocular oathology. To study the pathogenesis of onchocercdl’ker.7titis, invrstitqtors have either inoculated live Mf into the conjun&” or cornea of laboratory animals, or injected sohlble parasite antigens into the cornea1 stroma. This approach induces keratitis within days or weeks, as compared with years for human infection. In general, intraconjunctival injection with live Mf induced punctate keratitis, whereas direct intracorneal injection of Mf or soluble sntieens induced a more severe inflammation resembling’&lerosing keratitis. Many of these stndies are outlined in Table 1.
Investigation o. ‘nunune mechanisms underlying onchocercal keratitis (&Cc? -. ~ nctate 3 sclerosing) has been hampered by the difficulty in obtaining corneas from infected individuals. Many of the corneal samples are from end-stage disease, when the corneas are mostlv fibrotic. Studies have therefore used adjacent cohjuncti:al tissue, and have demonstrated abscesses compnsing dead Mf with eosinophils and T cells were also found to infiltrate the lymphocytes’“. conjunct& of patients, and resident cells were in II stati of activadon as measured by major histocomoabbilitv com&x (MHC) class II exoressioa”. Chan if nl. d&ted bterleukin 4 (IL-4) mRkA in conjunctivae from seven out of ,en ot these patients, but found no correlation with disease statu9, However, IL-4 and IL-5 production by peripheral blood mononuclear cells (PBMC) stimulated with parasite antigen were found to o,rrelate positively with ocular and dermal disease tD.0. Freedman, pers. commun.). These studies indicate that IL-r (which induces B-cell switch to IgE srcretion) and IL-5 (which stimulates eosinophil production) are likely to be involved in immunopathologj of orular onchocerciasis.
Primates as models Donnellv and co-workers have rcoorted a series of experirne& in which live Mf of either 0. oulwhrs or the cattle pawite 0. lirrmlis wcie inoculated into various sites ;n the eyes of cynomolgus monkeys”,‘“‘“. Animals injected intracomea!ly showed a minimal inflammatory response, even if they had been previously immunized”. The absence of corneal lesions in these singly challenged animals suggests that chronic invasion of large numbers of Mf is necessary for develooment of lesions. These results are consistent with’ human disease, but differ from sh-dies using guinea-pigs inoculated with live 0. licne’is Mf (see below). The prtmatc model rn”v be more useful or studying posterior disease, as qection of 0. liwnlis Mf into the vitreous or pusterior areas of the eye induces a pronounced inflammatory reqxmse comprising monw cyts and densely packed maws of eusinophils. Tiiese inflammatory lesions are often seen to surround Mf in the vilreous area, indicating a role for thee cells in parasite cleamnc@. Comparison of rainforest and savanna strains Duke and co-workers were among the first to use animal models to study the development of 0. ralo~d~rs
Reviews mediated keratiti+x. These investigators recovered live 0. oolvr~lrrs Mf from skin snigs of volunteers from either the savanna or the rainforest region of Cameroon. Microftlartae WlG3X-O) were then inoculated directly into the conjunctiva of mbbitsat several sites around the cornea. Although only a small percentage of the inoculated Mf invaded the cornea, about three times more savanna strain parasites than rainforest strain Mf were found to be present (O&4.5% vs O.l-2.4%). In addition, the savanna strain parasites induced a more intenae inflammation, characterized by punctate keratitis, comeal vascularization and occasional stromal keratitis. Histoloeical exam cat% of corneas showed that infiltration t? infiammatory cells, primarily eosinophi!s and lymvhocytes, was greater in rabbits in&&ted with- the savanna strein than with the minforest strain2’. Jnoculation of dead Mf directly into the cornea1 stloma YesuIted in relatively mild lesions wth no differences in clinical or Qath&Jgical reaction observed between the two stmins (kemtiiis did not develop if killed Mf were injected into the conjunctiva). The difference in virulence between these strains is therefore more likely to be clue to the invasive chamcteristics of ~~annn strain Mf than to intrinsic differences in Qrwasite rmtigens between the rtmins. This conclusion is also consistent with observations ot posterior eye disease induced after intravitreal and subretinal inlettion of live Mf into rabbits. Although thse animals develooed an inflammatorv resoonse. there was no differ&e between savanna and ramforest strains of the pamsite”~~~~. To determine the effect of pre-existing immune raponses on the dcvelopmcnt of kcratitis, rabbits were immunized intravenously with live Mf prior to intraconjunctival challenge. These animals displayed punctate kentitis one zlzy after injection compared with four days in unimmunized animals. lr addition to the accelerated appe.~nnce of &ease in immunized rabbits, the severity of keratitis was enhanced and contained a prominent neutrophil infiltmte”. In contrast to injection with live Mf, rabbits immunized intmvenously with dead Mf appeared to be tolerized to subsequent exposure to live Mf, which is a more typical outcome of intravenous injection. These animals had only minimal ocular pathology, and motile Mf were detected in the cornea up to 17 days after inoculation. Rodent models using Mf Given the difficulty in obtaining sufficient numbers of 0. WIVUIIW Mf from infected individuals, subsequent studies have used Mf recovered from horses or cattle. lntraconjuncttval inoculation of rabbits and guinea-pigs with 0. gettwusn Mf from cattle did not ip duce keratitis”. O~rl~ocwcn cervicalis Mf isolated from horses invade the comeal stroma of CSA/H micp after subcutaneous injection, but no inflammatory response was detected in these animals, even after repeated injection and treatment with diethylcarb amazine (DEW (E.R. James, per% commun.). In contrast to studies descrtbed above using 0. gtrtterosn and 0. ccrvicnlis, Mf of the caltIe parasite 0. licruk penetrate the cornea and induce tipacities resembling human punctate keratitia when inoculated intmconjunctivally into Hartley strain guineaPoronltologyTodoy.vol. I2. “0 7. 1996
pig+‘“. Histological examination of the lesions reveals foci of eosinophils and mononuclear cells together with local edema, which is similar to that reported for human onchocercte.sislJ.~. Repeated subconjunctival inoculation with live 0. limnlis Mf c.auses more rapidly aQ;2earing and severe pun&ate lesions, whereas sut.xtaneuus immumzation suppressed the development of keratitis after subconjunctival inoxul&ion. Sakla c: n1.z suggest that the immune response elicited by subcutaneoi~s immunization results in death of the Mf before they can penetrate the cornea. Although it is not known why the immune response to subconjunctival immunization would then result in eracc:bated pathology, it is possible that the Qamsites are not killed in the conjunctiva, but are able to penetrate the cornea and become the focus of an inflammatory response at that site. In contmst to observations with subconjunctivally iniccted Mf, direct injection of live Mf into the correal btroma of subcutaneously immunized guinea-pigs resulls in an intense keratitis, resembling scterosing keratitls in hilmans2”‘. These animals dwelop nea vascularizaticn and opacities covering up to half the cornea. The opacities are found to be associated with comeal edema and recruitment of eusinophils, neutrophils, lymphocytes and plasma cells. Keratitis is not induced unisss the animals either are previously sensitized or have rweived spleen cells from immunized animals prior to intracomeal inoculation, demonstrating thnt celluiar responses are required for develop ment of !.eratitiP’. Cornea1 inflammation also cormlatch with increased IgE levels both in sera and in aqueous humor, indicating that this antibody isotype is involved in mediating keratitis. The QESEmce of IgE and wainophils in this model is consistent with human disease manifestations and with production of IL-4 and IL-3 (Refs 40,411. The capacity for 0. limnlis Mf to induce keratitis in guinea-pigs, but not in cynomolgus monkeys, may be due to subtle anatomic differences in the corneas of Qrim.,teS and rodentsn. The investigators suggest that Bowman’s membrane, which underlies the comeal epithelium. is thick- in primates and humans than in rodents, and is relatively redstant to neovascularization and subsequent keratitis. Rodent models using 0. oolvwlus antigens The observations in human infection and in the animal models (described above) demonstrate that keratitis is induced after Mf havr ratered the cornea and died, releasing internal mtigens. Injection of 0. uolr~drrs antigens COvAgl directly into rhe cornea could then substitute for live Mf. Using this approach, unsensitized guinea-pigs and mice have a mild, inflammatory response with minimal stromal infiltrate, whereas animals receiving prior immunization with OvAg develop pronounced cornea, opaciticatton and neovasculartzatton26~“’ (Fig. 2). The inflammatory response is similar to that described above for live Mf. The composition of the cellular infiitrate was dependent on the adjuvant used during systemic immunization, as animals @en a single immunization of OvAg in complete Freuncls adjuvant prior to inb;lcomeal injection develoQ+d kemtttis in which neutrophils are present during tlw initial three days, and lymphocytes, plasma cells and 163
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histiocytes are present after seven dayrWq. In contrast, mice given repeated immunizations of OvAg in a squalene based adjuvant (with no mycobacterial antigens) prior to intracomeal injection develop a maximal response on Days 4-10, and the cellular infiltrate is uredominantlv eosinauhils (Fig. 3)“‘. The difference ‘m cellular in&ate b&wee” &he two models has yet to be determined. The role of CDl+ T cells and IL-4 Chakravartt and co-workers have shown that CD4’ T cells rather than CD8+ T cells are predominant in the corneas of A/J mice throughout the course of keratitis, implicating a role for these cells in the inflammatory responses’. On the basis of cytokine product:z, CD4* T cells can be grouped into T helper cell tyFe 1 CThl) and type 2 (Th2) subtypes. Thl cells produce interferon y (IFN-$, implicated in delayed-
type hypersensitivity reactions, whereas Th2 cells oroduce IL-4 and IL-5. which are reauired for mohuction of IgE and eos&mphils, respedively~~. A;described above, eosinophils are prominent in inflamed corneas of rabbits and guinea-pigs inoculated with live MPQ7.11 and IgE is present in the aqueous humor in infected guinea-pig+“. These observations suggest that induction of ThZ-cell-associated r~ponses are involved in development of kemtitis. A more direct assessment of the Th-cell response induced by 0. vnfvul~rs antigens has been determined by examining cytokine production by lymph node and spleen cells from BALB/c mice immunized subcutaneously with OvAg)‘. OvP.g-stimulated CD4+ T cells produce IL-4 and IL-5, but not IFN?, thereby demonstrating a predominant antigen-specific ThZcell response. Cytokine pmduction at the site of iiulamnation is determined by reverse tmnscription
Reviews PCR (polymerase chain reaction) using corneas removed at the peak cf the inflammatarv restw”se (ie. seven days after in&&wd injection). OvAg-injected corneas have elevated levels of CD4’ and CDEV T cells and IL-4 gene expression compared with unbjected corneas from the sane animals (Fig. 41, although uninjected corneas demonstrate CD8 expression. Consistent with in vilro cytokine production, IFN-y expression remains low, indicating that ThZ- rather than Thcell responses dominate at the site of inflammation. As IL-4 is required for development of Th2 cells, its role in 0. uoluzrlwmediated stromal keratitis has been determiwd in mice in which the iL-4 gene has been disrupted? In contrast to control C5781/6 and F2 mice, IL4-deficient mice develop significantly less keratitis than do control mice, thereby demonstrating an essential role for IL-4 in immunopathology”. Histological examination reveals that the cornea1 stromas of IL4-deficient mice are not edematous and contai” very few inflammatory cells (E. Pearlman, a:qxblished), suggesting that IL-4 may facilitate entry of inflammatory cells into the comeal stroma. Link between and keratitis I
,
Th2-cell
.
r CD4
Q
CD8
w
IFN-r
-
IL-4
a
HPRT
m
responses
.
ad on known functions of r&dent cells in the cornea, a sequence of immunwnediited events leading to keratitis has been propoSea (Box 11. Repeated or chronic exposure to OvAe induces a oredominant ThZcell ~&~nse sys&cally, with elevated serum IgE and eosinophilia, and IL4 and 1LSpmducing CD4f T cells. Injection of parasite antigen into the comeal stroma, or active penetration by live Mf, may stimulate cornea1 epithelial cells to produce inflammatory cytokinff such as IL-1 and tumor necrosis factor u (TNFn), which stimulate comeal iibmblasts (keratocytes) to release chemokima and enhance the expression of intercellular adhesion molecule-l (ICAM-1) and vascular adhesion molecule-l (VCAM-I) on vascular end&&l cells. IL-la and TNFa stimulate keratocyte pm&cticm of RANTES (regulated upon activation, normal T expressed and weted) and MCP-I (monocyte chemotactir protehW* which attract lymphuiy is and eosinophils in oilro. IL-la and TNF-a also stimulate integrin expression on human umbilical endothelial cells grmw in uitrd’, and are likely to have a similar effect on capillary venules. Upregulation of ICAM-I and VCAM-I on end&h&l cells may facilitate adherence and transmigration of wsinophils and OvAgspecific Th2 cells into the cornea1
Box 1. Predicted Sequence of Events Leading to Onchocercal Keratitis Svstemic rewonse ‘Repeated i;nmu”iratio” with parasite, 0. m,or,,r,s, anti&en (OvAg, selectively induces a ThZ-cell resplnse chronic infection in human disease). * OvAg-specific Th2 memory cells circulate through the vasculah~re.
*
(mimics
Immediate response at site of i+ctin” * Injection of parasite antigen int” the comeal stroma UTactive penetration of live Mf tigers release of inflammatory cytokines IL-I a”d TNF-u from cwneal epithelial cells. . IL-1 and TNF-a induce stromal f&v&lasts (kemtacvtcs, to ~roducc chemokines which attract Ivm~hocvm and I eosinophils. Inflammatory cytokines als” stimulale expression of molecuuln on capillary ve”“les. * MHC class LIpositive La”gerha”s ce11smigrate from the limb”.? to the cornea, stnnna. * A”giogenic factors are released. I
*
.
.
adhesion
Infiltration of inflammatory cells . OvAS-spedfic Th2 cells and eosinophils b,“d m capillary endothehal cells and migrate to the romeal stroma. * Class II paitiw La”gcrh.,“s EclLspiesent atigen to T cells. . Activated Th2 cells secrete cytukine and chemokines, leading tc further recmihnent of inflammatory cells. . IL-4 sti”ukIte5 further Thz-cell pralifemtio”. ThZ-cell-mediated inflammatory response * Eosinophils degranulate, releasing eosinophil major basic protein and o,her mediators into the strcona, matrix. . Release of i”flammat”ry mediators disrupts the co!lage” fibril arrangemm~ * Carneal edema ensues, resulting in loss of transparency. * Further release of angiogrnic factors stimulates mmeal “mvascularizati”“.
and comeal endothelial cell function.
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injury
stroma, where they may migrate to the site of by following a chemokine gradient. Further stimulation of Th2 cells at this site would require antigen presentation. As nnther fibroblasts nor cornea1 epithelial cells express MHC class II moldepends on migrstion of ecules, antigen presentation either monocytes from the circulation or, more likely, migration of Langerhans dendritic cells. Langerhans cells are potent antigen-presenting cells which reside in the conjunctive and cornea1 limbus, and which migrate to the central cornea in response to injury or infection’. Antigen presentation activates OvAgspecific Th2 cells, leedins to IL-4-mediated T-cell proliferation and productmn of IL-5 and RANTES, and rasinopbil recruitment. Degranuhtion of eosinophils would release mediators, including ma@ basic protein, eosinophil cationic protein and eosinophil peroxidase. These can be expected to disrupt stromal tibril arrangement either directly (by interacting with the collagen and proteoglycan). or indinxtly (by impeding the function of the Na’ /K*-ATl’ase-depadcnt pump in corncal endothelial cells, thereby increasing the hydration level of the cornea1 stroma). This scenario is con&tent with the observed comeal edema. In either case. verturbathrt: cf the collagen fibril arrangement will &It in loss of transparency and a clinical picture of cornea1 op.xification~ The factors underlying cornea1 neovasculatiation in onchocercal keratitis have yet to be determined. Given the variety of cell types present, it is likely that an array of angiogenic factors are involved, iwILding IL-I, IL-Z, IL-S, TNF-ol and tmnsforming growth factor p (TGF-PPH”y. In addition to production of angiogenie factors, the inflammation may have an indirect role in disrupting the balance of angiogenic and angiostatic factors that is essential for the maintenance of the avascular nature of the normal cornea. Other factors, such as autoimmune responses, may also contribute to the observed pathology.
Relation to human disease Although the predicted sequence of events is derived primarily from observations in animal mod&, there are at least three features that are common to human disease: (1) the requirement for prior sensitization (as a result of either chronic infection or prior immunization), which results in an appropriate cellulx response; (2) the presence of parasite antigens in the cornea1 stroma either after invasion of live Mf or by direct injection of parasites or parasite antigen; and (3) development of a sustained inflammatory response in the corn& Worna. A more complctc understanding of these events may suggest a rationale for immune-based intervention which could potentially limit development of 0. vulwdus-mediated ocular disease.
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Lymphatic Filariasis: What
Mice Can Tell Us R.A.
Law-ence m.&nisms used by the parasite will make it possible to tareet strateeies for immune intervention. The use of an&al mod& is an essential link in our understanding of immunity to filartasis, because it is only in these systems that we can combine knowledge of the immune rejponse with knowledge of the duration of infection, the developmental stages present, the infection dose and the site of infection. An ideal animal model of lymphatic filariasis would ha&our the full developmental cycle and dismimplay similar pathology to humans in addition icking the immunological characteristics of human infection. In the absence of a convenient laboratory model with these attributes, a surprising amount has been learnt from the study of short-term infections in mice. With the advent of genetically manipulated mice in which specific pathways of the immune Esponse have been deleted, the importance of precisely defined immune parameters can be assessed in &xctective immunity, parasite sup and host pathology. This review draws together our current knowledge of tilariasis in the mouse, and highlights areas in which further research can provide answers to the major questions in lymphatic filariasis as well as give new direction to human studies
to
Iid
and to determine the CBUSBof the patholdgical res~cmsesseen in others. Furthermore, the parasite SUP v&es at the heart of the Immune system &I extended periods of time. Identification of the immune evasion
The filarial parasites Brrrgin sp. and W. bnncrofC are transmitted by mosquito vectors. Infective stage larvae (L3) initiate infection when the mosquito feeds upon the human host. The larvae migrate to the lymphatic vessels and mature into adult male and female worms. After mating, the female worms prcduce