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Characterization of local immune response against lungworms innaturally infected sheep

M. Yavuz Gulbahar a,*, William C. Davis b, Murat Yarim a, Tolga Guvenc a, Sinasi Umur c,Yonca B. Kabak a, M. Onder Karayigit a, Y. Emre Beyhan c

a Department of Pathology, Faculty of Veterinary Medicine, University of Ondokuz Mayis, Kurupelit, Samsun, Turkeyb Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, United Statesc Department of Parasitology, Faculty of Veterinary Medicine, University of Ondokuz Mayis, Kurupelit, Samsun, Turkey

1. Introduction

Lungworms cause serious diseases that lead to death inyoung animals, but they may also reside in the lungs withlittle apparent effect on the host (Jones et al., 1997). Thelungs are at the crossroads of parasitic migrations, and thenumerous parasites that pass through the lungs causevarious degrees of damage according to the nature andintensity of the host–parasite interaction. Some parasiteslocalize in the airways and the parenchyma of the lungs(Dungworth, 1993).

Parasitic diseases of the ovine lung appear to becommon in Turkey, and Cystocaulus ocreatus, Neostrongylus

linearis, Dictyocaulus filaria, Muellerius capillaris, andProtostrongylus spp. are found to be the common nema-todes that parasitize the lungs of sheep in the Samsunprovince. In addition to infections associated with a singlespecies of parasite, mixed infestations of more than onespecies in the same lung have been also encountered(Cetindag, 1993).

Immune response against helminth parasites has beenstudied in various parasitic diseases of ruminants, such ashaemonchosis (Gorrell et al., 1988a,b; Gill et al., 1993;Mansfield and Gamble, 1995; Amarante et al., 1999; Perezet al., 2008), ostertagiosis (Almeria et al., 1997a,b),trichostrongylosis (Gorrell et al., 1988a,b), hydatidosis(Bauder et al., 1999; Sakamoto and Cabrera, 2003), andfasciolosis (Perez et al., 2002). The cellular reaction to thevarious developmental stages of lungworms in sheep

Veterinary Parasitology 160 (2009) 272–278

A R T I C L E I N F O

Article history:

Received 2 September 2008

Received in revised form 5 November 2008

Accepted 10 November 2008

Keywords:

Lungworm

Sheep

Local immune response

Immunohistochemistry

Lung

A B S T R A C T

This study describes the immunohistochemical and histochemical phenotypes of

inflammatory cells in sheep lungs infected with lungworms. A total of 20 naturally

infected sheep lungs were used. Protostrongylus spp., Muellerius capillaris, Neostrongylus

linearis, and Cystocaulus ocreatus were the chief organisms determined from such lesions,

which were of a chronic nature. All the lungs had many developmental stages of the

parasites and a similar inflammatory response, which included numerous mast cells,

eosinophils, T cells, B cells, dendritic cells, and macrophages. In the bronchial and

interstitial tissues, the inflammatory cells were dominated by MHCII, CD1, CD4, CD5,

CD14, CD21, IgM, and CD172a positive cells, whereas CD2 and WC1 positive cells were

detected less. The data provided additional evidence that subsets of inflammatory cells

were included within ovine lungs infected with lungworms; however, understanding the

entire immune-response process and development of resistance to lungworms in sheep

remain to be clearly elucidated.

� 2008 Elsevier B.V. All rights reserved.

* Corresponding author at: Department of Pathology, Faculty of

Veterinary Medicine, University of Ondokuz Mayis, Kurupelit, 55139

Samsun, Turkey. Tel.: +90 362 3121919x2818; fax: +90 362 4576922.

E-mail addresses: mygulbahar@hotmail.com, myg64@omu.edu.tr

(M.Y. Gulbahar).

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsevier .com/ locate /vetpar

0304-4017/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.11.012

Author's personal copy

include the mobilization of eosinophils, mast cells,macrophages, and multinucleated giant cells, which fillthe alveoli, alveolar septa, and terminal bronchioles. Thereis a mild fibrous thickening of the alveolar septa, withmany lymphocytes embedded in the septa, and around theblood vessels and bronchioles (Dungworth, 1993).Although there are a few experimental studies associatedwith the immune response against lungworms, includingP. rufescens in sheep (Mansfield and Gamble, 1995; Collieet al., 2001) and D. viviparus in cattle (Eysker et al., 2001;Johnson et al., 2005), the detailed immunohistochemicalcharacteristics of the local cellular response againstlungworms in sheep have not been investigated.

The purpose of this study is to characterize the localhost response, both immunohistochemically and histo-chemically, in natural infections of sheep with lungworms.

2. Materials and methods

2.1. Collection of lung samples and parasitological

examination

A total of 20 lungs with parasitic pneumonias wereobtained from adult sheep at local abattoirs situated inSamsun, Turkey. Seven lung samples from uninfectedanimals served as controls. Macroscopical findings withreference to the localization and features of the lesions onthe lungs infected with lungworms were recorded.

2.2. Histochemistry

Tissue specimens were obtained from the lungs withparasites, confirmed by parasitological examinations, andsubsequently fixed in buffered formalin. Tissues wereembedded in paraffin, sectioned at 5-mm thicknesses, andstained with (a) hematoxylin and eosin for globularleukocytes and eosinophils and (b) 1% toluidine blue formast cells.

2.3. Immunohistochemistry

Additional unfixed lung specimens were used forimmunohistochemistry. Cryostat sections were sectionedat 5 mm, mounted onto organosilane (3-aminopropyltriethoxysilane)-coated glass slides, and immediately fixedfor 8 min in ice-cold absolute ethanol. Frozen sectionswere prepared using a commercial streptavidin–biotinperoxidase system (SABC) (Zymed Laboratories, Inc., CA).The frozen sections were immediately air-dried using ahair dryer (cold air) to avoid autolysis of cells and kept at�20 8C. All sections were preincubated in a blockingsolution containing 10% goat nonimmune serum (ZymedLaboratories, Inc., CA) at room temperature (RT) for 10 minto block nonspecific binding of the second-step antibody(Zymed Laboratories, Inc., CA). Sections were treated withprimary antibodies overnight at 4 8C and rinsed withphosphate-buffered saline, pH 7.4, (PBS) at RT. Themonoclonal mouse antibodies used in this study and theirspecificities included:

Anti-ovine major histocompatibility complex (MHC)Class I (H58A, MHCl I, diluted 1/50) for most nucleated

cells, with the exception of neural cells; anti-ovine MHCClass II (TH14B, MHCl II, diluted 1/50) for B cells, activatedT cells, and dendritic cells (DCs); anti-ovine CD1 (TH97A,diluted 1/25) for thymocytes and a subset of DCs; anti-ovine CD5 (ST1, diluted 1/25) for all T cells and a subset of Bcells; anti-ovine CD2 (MUC2A, diluted 1/25) for ab T cells;anti-ovine CD4 (GC1A, diluted 1/25) for T-helper cells;anti-ovine CD8 (CACT80C, diluted 1/25) for T-cytotoxiccells; anti-ovine WC1-N2 (BAQ4A, diluted 1/50) fordetection of a major subset of gd T cells present inruminants; anti-B cells (BAQ44A, diluted 1/25; and GB26A,diluted 1/25); anti-ovine CD21 (GB25A, diluted 1/25) formature B cells and follicular DCs; anti-ovine sIgM(PIG45A2, diluted 1/50) for B cells; anti-ovine CD14(CAM36A, diluted 1/25) for monocytes and macrophages;anti-ovine CD172a (DH59B, diluted 1/50) for granulocytes,monocytes, macrophages, and a subset of DCs. Themonoclonal antibodies (mAbs) used in this study wereobtained from the Monoclonal Antibody Center, Washing-ton State University, Pullman, WA (Table 1). The sectionswere treated with biotin-conjugated second-step antibody(Zymed Laboratories, Inc., CA) for 10 min at RT andsubsequently rinsed in PBS at RT. For inactivation ofendogenous peroxidases, the sections were incubated in0.3% H2O2/methanol for 60 min. The sections were nextrinsed with PBS at RT and made to react with thestreptavidin–biotin-peroxidase complex (Zymed Labora-tories, Inc., CA) for 10 min at RT. After another washingwith PBS solution, the sections were further incubatedwith diaminobenzidine (DAKO, Carpinteria, CA) for 15 min,postfixed with 10% formalin, followed by washing withdeionized water, and finally counterstained with Harris’shematoxylin.

Negative controls were prepared both by omitting theprimary antibodies and by replacing them with one of thefollowing: PBS, normal rabbit serum, or unrelated mousemonoclonal antibodies.

2.4. Evaluation of histochemistry/immunohistochemistry

The cells in a total of 10 randomly chosen fields, of areas0.144 mm2, per lung were counted. Photomicrographs ofboth control and infected lungs were obtained for each celltype at 400�magnification by two pathologists (MYG andMY in the author line). The areas of the lung sections wereselected from two different anatomical regions: (1)bronchiolar tissues (including peribronchiolar, intrae-pithelial, and intraluminal regions) and (2) interstitialtissues (interalveolar, intra-alveolar, subpleural, and inter-lobular regions). The globular leukocytes were counted in10 randomly selected areas of sections from the bronchi/bronchiole of the lungs. The cell counts were expressed as apercentage of the total cells per lung and scored as follows:�, 0%; +, <5%; ++, 6–10%; +++, 11–50%; ++++, >50%.

3. Results

3.1. Gross and parasitological findings

Typical lesions were slightly raised multiple sub-pleural nodules, which were 0.5–2.5 cm in size and tan to

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gray-greenish in appearance, localized to the consolidatedareas of the lung, especially in the dorsal regions of thecaudal lobes of the lungs. Protostrongylus spp. and M.

capillaris were identified in such lesions. Cross sections ofthe nodules showed fine, white, hair-like worms. Smallerdark and firm nodules were 0.5–1 cm in size. This type ofnodule contained N. linearis and C. ocreatus.

3.2. Histochemistry and immunohistochemistry

In the infected lung sections, there were variousdevelopmental stages of the worms, including adultworms, larvae, or embryonating ova within the alveoli,alveolar ducts, bronchioles, and bronchi. Adult wormswere detected especially in large bronchi, whereas the eggsand larvae were found in the alveoli. Varying degrees ofcellular reactions to these parasites were observed withinthe same sections of the lung tissue. Perivascular andperibronchiolar lymphoid hyperplasia extended to largeareas of the lung parenchyma, with various inflammatorycells such as lymphocytes, plasmacytes, and macrophagespresent within both the alveolar septa and the lumens ofthe bronchiolar and alveolar tissues. There were fibrousthickenings of the alveolar septa (with lymphocyteinfiltration), around the blood vessels, bronchi, andbronchioles, and between the bronchial glands. In someregions, the parasites in the alveoli were encapsulated withdense fibrous tissue. Some of the alveolar septa weredisrupted by the parasites and many macrophages withclear nuclei and a few multinucleated giant cells wereincluded. The results of histochemistry and immunohis-tochemistry are summarized in Table 2. There was nocorrelation between the species of parasite and theassociated inflammatory reaction in infected lungs.

Mild-to-intense foci of scattered or grouped eosino-phils and mast cells were detected. A significant numberof mast cells appeared in the perivascular and peribron-chial tissues, whereas many eosinophils were noted inthe alveolar septa surrounding the worms and within thebronchial lumina. The globular leukocytes with typicaleosinophilic cytoplasmic granules were seen within theepithelial tissues of the bronchi/bronchioles in theinfected lungs, but these were not observed in thecontrol lungs.

MHCI expression was detected in the inflammatory cellsand most of the cellular components of the lung, includingthe epithelium of the alveoli and bronchi/bronchioles, andthe endothelium of blood vessels. There was no differencebetween the infected and control lungs with reference to thecellular components expressing MHCI. However, in theinfected lungs, immunostaining for MHCI was more intensethan in the controls because of the mass of inflammatorycells. MHCII immunostaining showed lymphocyte infiltra-tion around the peribronchi and in the interalveolar tissues.Moreover, DCs, macrophages, and multinuclear giant cellsalso expressed the MHC II antigen (Fig. 1). MHC IIimmunostaining was more intense in infected lungs thanin control lungs. CD1 immunostaining showed clustered andscattered cells in the perifollicular areas of the hyperplasticlymphoid cuffs, which extended to the interstitium (Fig. 2).Although the cells that were immunostained for CD1 had

Table 1

Monoclonal antibodies used in this study.

Antigen Antibody Ig isotype Dilution Reactivity

MHC Cl I H58A IgG2a 1/50 Most nucleated cells, with exception of neural cells

MHC Cl II TH14B IgG2a 1/50 B cell, activated T cells, dendritic cells

CD1 TH97A IgG2a 1/25 Thymocytes, subset of dendritic cells

CD5 ST1 IgG2a 1/25 All T and a subset of B cells

CD2 MUC2A IgG2a 1/25 ab T cells

CD4 GC1A IgG2a 1/25 T-helper cells

CD8 CACT80C IgG1 1/25 T-cytotoxic cells

WC1-N2 BAQ4A IgG1 1/50 Major subset of gd T cells

B BAQ44A IgM 1/25 B cells

CD21 GB25A IgG1 1/25 Mature B cells and follicular dendritic cells

B GB26A IgM 1/25 B cells

SIgM PIG45A2 IgG2b 1/50 B cell

CD14 CAM36A IgG1 1/25 Monocytes, macrophages

CD172a DH59B IgG1 1/50 Granulocytes, monocytes, macrophages, dendritic cell subset

Table 2

Semiquantitative scoring of inflammatory cells stained by histochemistry/

immunohistochemistry in ovine lungs naturally infected with lung-

worms.

Control lungs (no: 7) Infected lungs (no: 20)

Ba I B I

Mast cells +b + ++ ++

Eosinophils � � ++ +++

GLc � � + �MHC Cl I ++++ ++++ ++++ ++++

MHC Cl II +++ ++ ++++ +++

CD1 ++ + +++ ++

CD5 +++ ++ ++++ ++

CD2 ++ ++ ++ ++

CD4 ++ ++ +++ ++

CD8 ++ + ++ ++

WC1-N2 + + + +

B (BAQ44A) ++ ++ +++ ++

CD21 +++ + +++ +++

B (GB26A) ++ + +++ +++

SIgM + + +++ +++

CD14 + + ++ +++

CD172a +++ ++ +++ ++++

a B = bronchiolar tissues, I = interstitial tissues.b Score of inflammatory cells (�) = 0%, (+) = �5%, (++) = 6–10%,

(+++) = 11–50%, (++++) = �50%.c Mean number of globule leukocytes in bronchiolar epithelium of all

lung.

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Figs. 1–12. (1) MHCII+ lymphocytes, dendritic cells (arrowhead) and macrophages (arrows) around the bronchiol plugged with some parasites (asterisk).

SABC method, hematoxylin counterstain. (2) The dispersed or clusters of CD1+ lymphocytes among the coiled larvae in the alveoli. SABC method,

hematoxylin counterstain. (3) CD5+ lymphocytes within the lymphoid cuff around the parasite larvae in the alveoli. SABC method, hematoxylin

counterstain. (4) Numerous dispersed CD4+ T cells within the lymphoid cuff. Note an embryonating ovum of parasite (arrow) in the alveoli. SABC method,

hematoxylin counterstain. (5) Less numbers of CD8+ T cells within the lymphoid infiltration around the parasites (arrow) compared with numbers of CD4+ T

cells in (4). SABC method, hematoxylin counterstain. (6) Some WC1+ T cells dispersed lung parenchyma infected with parasite. Arrows indicate

embryonating ova. SABC method, hematoxylin counterstain. (7) CD21+ B cells in the center of lymphoid follicle, which dispersed in the septum of alveoli.

Note various developmental stages of parasites in the alveoli. SABC method, hematoxylin counterstain. (8) Scattered B cells immunostained with antibody,

BAQ44A within the lymphoid follicle around parasite (arrows) in the alveoli. SABC method, hematoxylin counterstain. (9) IgM+ immunostaining of linear

pattern on the some adult parasite (arrow) and coiled larvae (arrowhead) in the hyalinized tissue. SABC method, hematoxylin counterstain. (10) Numerous

CD14+ macrophages within alveoli (arrows) and around (arrowheads) the parasites. SABC method, hematoxylin counterstain. (11) CD172a+ granulocytes,

possibly eosinophils within/around bronchiol and some macrophages (arrows). Asterisk indicates a coiled larvae. SABC method, hematoxylin counterstain.

(12) Numerous CD172a+ macrophages and dendritic cells among the various developmental stages of parasites in the alveoli. SABC method, hematoxylin

counterstain.

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mononuclear morphology in general, few cells showeddendritic morphology.

Infiltration with CD5+ T lymphocytes was a commonfinding in infected lungs. The majority of CD5+ cells werefound in the hyperplastic perivascular/bronchiolar lym-phoid cuffs and lymphoid infiltrations surrounding theparasites and, to a lesser extent, in individual lymphocytesscattered throughout the interstitium. Intraepithelial CD5+cells were also common in the bronchi/bronchioles (Fig. 3).CD2+ cells had no predilection for localization in the lungtissues. The total percentage of CD2+ cells in control andinfected lungs did not change.

The percentage of CD4 cells was highest in circumfer-ential mantles around the lymphoid follicles, but they werealso found scattered throughout the interstitial spaces. CD8-expressing cells were stained predominantly in the periph-ery of peribronchial cuffs and individual cells that werescattered throughout the interstitium. The parasitized areasincluded more CD4+ and CD8+ cells than the other areas. Inthe bronchial tissues, the percentage of CD4-expressinglymphocytes had a greater score than the CD8-expressingcells. CD4+ and CD8+ cells were found occasionally inintraepithelial and intraluminal locations (Figs. 4 and 5).

A few gd-T lymphocytes expressing the WC1-N2antigen were noticed both within the lymphoid nodulesassociated with the bronchiolar tissues and within thealveolar septa as dispersed individual cells (Fig. 6). The B-cell marker CD21 was detected by the presence of stronglystained cells primarily within the center of lymphoidfollicles. The DCs scattered among the parasites were alsoimmunostained for the CD21 antigen (Fig. 7). In infectedlungs, the bronchial cuffs had an increased percentage of Blymphocytes, as detected by SIgM and other B-cellmarkers, BAQ44A (Fig. 8) and GB26A, which were locatedmainly in the center of the lymphoid follicle. B cells wereobserved to be less in proportion in the interstitial tissues.SIgM immunostaining was also found on the parasitaryelements and in the fluid leakage in alveolar and bronchiallumina. Immunostaining of the parasites for SIgM antibodyshowed a linear pattern (Fig. 9).

CD14 immunostaining was detected in large cells withpale nuclei and abundant cytoplasm, which occur mainlyin contact with the parasitic elements. CD14+ macro-phages were localized as clusters in the alveoli and asoccasional cells in the peribronchial, perivascular, andsubpleural areas (Fig. 10). Numerous CD172a positive cellswere present in infected lungs. Inflammatory cells withthree different morphologies were detected by the CD172aantibody. Granulocytes were found within the interstitialtissues and the bronchial/bronchiolar lumina. The percen-tages and immunostaining properties of the CD172a+macrophages, with large cytoplasms and pale nuclei, weresimilar to the CD14 immunostained cells in both theinfected and control lungs. Small populations of CD172a+DCs were detected near the airways and throughout theinterstitial tissues (Figs. 11 and 12).

4. Discussion

The cellular reaction to lungworm infection reflects thedevelopmental stage of the parasite and the state of

resistance of the host; the infection nodules are formed bymasses of adult worms, embryonated eggs, and coiledlarvae (Dungworth, 1993). In this study, the inflammatoryreaction generally is of a chronic nature and includesdiffuse or focal lymphoplasmacytic and eosinophilicinfiltration, mastocytosis, and fibrosis. Because all thesamples show every stage in the development of theparasite and because the lesions are characterized bychronic inflammation, the association between the lesionsand parasites has not been determined. Moreover, thisstudy shows that lungs with mixed infestations alsoexpress an immune response similar to that of single-parasite infestation.

There have been many studies about the immuneresponse in ruminant models to some gastrointestinalnematodes, such as haemonchosis (Gorrell et al., 1988a,b;Perez et al., 2008; Balic et al., 2000) and trichostrongylosis(Gorrell et al., 1988a,b). In experimental infection with theHaemonchus contortus larvae in sheep, the early immuneresponse is characterized by activation of eosinophils,CD4+ cells (coexpressing the MHCII and CD25 antigens),and gd-TCR, WCI+ T cells, and B cells, whereas infectionwith the adult parasite stages shows only an increase innumber of mast cells as the local response (Balic et al.,2000, 2002). Similarly, the cellular profiles in the abomasalmucosa of goats infected with H. contortus are evident fromthe T-cell subsets such as CD2, CD4, and gd T cells (Perezet al., 2008). Almost equal numbers of cells expressing CD4,CD5, and CD8 antigens have been detected in H. contortus-infected sheep (Gorrell et al., 1988a,b). The immuneresponse to lungworms has been identified, whichincludes the activation of substantial numbers of eosino-phils, mast cells, many T-cell subsets expressing CD4, CD5,and CD8, and B cells especially expressing CD21 and IgM, inaddition to many DCs expressing MHCII and monocyticmacrophages expressing CD14 and CD172a. The presentstudy shows a smaller proportion of CD2+ and WC1+ Tcells, but a large proportion of CD4+ T cells. Thegastrointestinal studies of the nematodes show increasedproportions of CD2 and WC1+ T cells (Perez et al., 2008;Balic et al., 2000, 2002). gd-T cells are believed to play arole in the first line of defence as effector cells andpotentially play a significant role in regulation of theimmune response that is restricted by the MHC andmediated through the actions of ab-T cells (Born et al.,1999). Ovine ab- and gd-T cells are thymus-dependentduring ontogeny, and these peripheral T-cell subsets havestrikingly different capacities for self-renewal and expan-sion after different antigenic stimulations (Hein et al.,1990). Following infection with Ostertagia ostertagi incattle, IgM, WC1, and IL2R+ cells are comparable topreinfection levels 2 months after the infection (Almeriaet al., 1997b). The differences in the immune responseamong the studies may have arisen from factors such asthe duration of infection, different mucosal immunities,and age of the animal or species of parasites.

DCs play a pivotal role in antigen capture and providingthe signals that determine the responses of effector T cells.The ovine respiratory tract contains a widespread popula-tion of DCs in airways and parenchyma, and they expressdifferent DC markers. This occurrence may represent two

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distinct populations of DCs or may represent DCs atdifferent stages of maturation (Fach et al., 2006; McNeillyet al., 2006). In this study, DC subsets expressed generallyby MHC II and CD21, and less frequency by CD1, have beenfound, suggesting that they may have a crucial role in theimmune response to lungworms in sheep. Moreover,widespread populations of DC subsets have been observedusing MHC II antibody.

After a challenge-infection of immunized sheep withgastrointestinal nematodes, two types of immuneresponses have been proposed as follows:

(a) the rapid rejection of incoming larvae, associated withchanges in globular leukocyte population, but nomobilization of the local immune system,

(b) the delayed rejection of the parasite with dramaticchanges in the local immune system when thechallenging larvae reach their tissue niche (Balicet al., 2002).

When the infection intensity decreases and the numberof globular leukocytes drops below a critical level forimmediate larval expulsion, the larvae will be able to reachtheir tissue niche and therein generate a pronouncedinflammatory response, characterized by the recruitmentof large numbers of eosinophils and Th2 cytokines againstgastrointestinal nematodes (Meeusen et al., 2005). In thepresent study, although it has not been ascertainedwhether the sheep have been immunized previously, allcases are suggested to show the delayed rejection phase ofthe parasites, characterized by tremendous local cellularimmune response, including mobilization of mast cells,eosinophils, MHCII+ T and MHCII DCs, CD4+ and CD8+ Tcells, CD14+ and CD172a+ cells, with no clear increase inthe numbers of globular leukocytes compared with thecontrols. A previous histochemical study shows that ovinebronchopulmonary globular leukocytes are abundant inthe respiratory tracts of most sheep showing parasiticlesions in their lungs; however, they are absent in someparasitized sheep (Mahmoud and Pirie, 1982). In goats,globular leukocytes belong to the gd-T cell subset, which isa cell population different from the WC1 positive gd-T cells(Konno et al., 1995); however, there is no informationavailable about ovine globular leucocytes also being a gd-Tcell subset, which remains to be determined. Althoughmechanisms about the recruitment or existence ofglobular leukocytes in the lungs of parasitized sheep havenot been elucidated, this recruitment may be associatedwith the duration of inflammation in the lung. Inruminants, recent studies related to the immune responsetoward helminth parasites have been conducted mostly ongastrointestinal nematodes. The pulmonary cellularresponse to lungworms and the inflammatory course ofinfection has not been well characterized in sheep. Afterhelminth infection in mice, Th2 immunity does increase IL-5 production, which greatly enhances the recruitment ofeosinophils to the lung (Culley et al., 2002). Eosinophilsand mast cells are the major effector cells typicallyassociated with helminth infections. Tissue mast cellsappear uniformly scattered in the tissues, whereaseosinophils show a directional migration toward the

parasitic target (Balic et al., 2000). Many mast cells havebeen identified in uninfected lungs, but not eosinophils.The infected lungs in sheep contain scattered or clusteredmast cells and eosinophils throughout the lung tissues. Inhumans, pulmonary mast cells have been found to reactwith the CD172a antibody (Ghannadan et al., 2002). In thepresent study, CD172a immunostaining also confirms theabundant numbers of eosinophils, and possibly mast cells,in bronchial and interstitial tissues. Additionally, eosino-phils serve as the major effector cells, inducing tissuedamage and dysfunction by releasing toxic granularproteins and lipid mediators (Rothenberg and Hogan,2006). The immune response to lungworms is detrimentalto the host, and protostrongylid infections are known toprogress with the age of sheep, accompanied by massiveinflammatory response to the adult parasites (Mansfieldand Gamble, 1995; Cabaret et al., 1980). The presence ofabundant inflammatory cells in the lung parenchyma ofinfected sheep alone does not appear to be sufficient for aprotective response; probable additional factors that maybe required for resistance to the lungworms need to bedetermined. Additional studies are needed for furtherelucidation of both the immune response and thedevelopment of resistance to lungworms in sheep.

Acknowledgements

The authors are particularly grateful to Washington StateUniversity’s Monoclonal Antibody Center for donating themAbs for this study.

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