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    Gross and microscopic aspects of the lungs observed in animals infected with S. venezuelensis (left) and infected-treated with dexamethasone (right). On Day 1, scarce hemorrhagic foci containing larvae are seen in the alveolar spaces of infected animals (inset: larva). A similar aspect is observed in infected-treated animals (arrows). The hemorrhagic foci are more prominent on Day 3 in both groups, showing intra-alveolar proteinaceous exudates, inflammatory cells, larvae (arrow), and cuticle fragments. This aspect is more severe in the infected-treated group, with confluent hemorrhagic foci. Larvae can be seen inside the rectangle. On Day 5, most of the hemorrhage is now composed of hemosiderin-laden macrophages that give a tan appearance to the pulmonary surface. Note the granulomatous aspect of the inflammatory infiltrate in the alveolar parenchyma (arrow). These aspects were not observed in the infected-treated group. The inflammation is still hemorrhagic, with prominent exudates and cuticle fragments. However, no larvae or granulomas are observed at this day in this group. After 7 days, the granulomas are well developed, composed by macrophages and eosinophils, and can be seen in the pleural surface of infected animals. The infected-treated group shows macrophage agglomerates without signs of granulomatous organization (arrow). On Day 14, the granulomas are scarcer in the alveolar parenchyma in the infected group, whereas in the infected-treated group, free larvae are seen in the alveolar parenchyma intermixed with hemosiderin-laden macrophages without granuloma arrangement. On Day 21, only residual granulomas are seen in infected animals and, in the infected-treated group, dispersed hemosiderin-laden macrophages are seen in the alveolar parenchyma. (H&E; original magnification, ×200; inset, ×640). Scale bars = 4 mm and 50 μm. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Eosinophilic infiltrate and mast cells (not seen in this stain) in the axial septum in controls, S.v.-infected animals, and infected-treated with dexamethasone. The most prominent eosinophilic infiltrate can be observed on Day 3 in the infected group. This infiltrate diminishes through Days 5 and 7 of the experiment. The infected-treated group shows a behavior similar to the eosinophilic infiltrate in the axial septum, although less prominently. The controls show no eosinophilic infiltration of the peribronchovascular tissue. (HE; original magnification, ×400). A, Mast cell distribution in the axial septum in all groups. Note the increase in mast cells on Days 3 and 14 after infection in the infected group, whereas the infected-treated group shows no increase in mast cells. B, Eosinophil distribution in the axial septum in all groups. Data are expressed as mean ± SEM. *P < 0.001, #P < 0.01, †P < 0.05. Scale bars = 50 μm. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Granulomatous formation in the alveolar parenchyma in S.v.-infected animals (left) and infected-treated with dexamethasone (right). The panel shows granulomas on Days 5, 7, and 14 in the infected group, composed by eosinophils and macrophages entrapped in a well-developed reticular fiber network, arranged concentrically. Note the presence of a larva in the center of the granuloma (arrow) at the fifth day after infection. In contrast, no evident reticular fiber organization was seen between the agglomerated macrophages in infected-treated animals. (HE and Gomori reticulin stain; original magnification, ×400). A, Eosinophils counted in the granulomas or inflammatory agglomerates in infected-treated animals. B, Volume fraction (%) of macrophages in the alveolar parenchyma. C, Reticular fiber densitometry (%) in the granuloma or around agglomerated macrophages in infected-treated animals. Data are expressed as mean ± SEM. *P < 0.001, #P < 0.01, †P < 0.05. Scale bars = 50 μm. This figure appears in color at www.ajtmh.org.

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Interference of Dexamethasone in the Pulmonary Cycle of Strongyloides venezuelensis in Rats

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  • 1 Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil; Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil; Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

The aim of this study was to investigate the interference of a daily treatment of dexamethasone in the pulmonary cycle of Strongyloides venezuelensis infection in rats. Three principal effects were found: 1) increased alveolar hemorrhagic inflammation provoked by the passage of larvae into alveolar spaces; 2) significant decrease of eosinophil and mast cell migration to the axial septum of the lungs; and 3) impaired formation of the reticular fiber network, interfering with granuloma organization. This study showed that the use of drugs with immunomodulatory actions, such as dexamethasone, in addition to interfering with the morbidity from the pulmonary cycle of S. venezuelensis infection, may contribute to showing the mechanisms involved in its pathogenesis.

INTRODUCTION

Strongyloides spp. are nematode parasites, generally distributed in warm, moist areas because such climates are suitable for the survival of the larval stage.1,2 This worm has a complex life cycle (in humans or animals), where the larvae penetrate the skin and travel through the bloodstream to the lungs, where they break into the alveolar spaces, ascend the respiratory tree, are swallowed, and continue their development in the small bowel.3 The obligate pulmonary phase of the life cycle typically occurs within hours after infection and lasts only a few days before the worms migrate to the intestine, where they develop into adults.4

The immune response observed in nematode infection and asthma raises obvious questions about the relationship between helminth infections and allergic disorders, especially in the pulmonary parenchyma. In both, activated Th2 lymphocytes orchestrate the inflammatory cascade, communicating with the two primary effector cells, the mast cell and the eosinophil, by release of interleukins 4 (IL-4) and 5 (IL-5).5 Although anatomo-pathological aspects of the alterations to the airways are well defined in the asthmatic process, little is known about the consequences of the passage of larvae in the pulmonary capillaries in nematode infections.

The objective of this study was to better characterize the pulmonary phase of a nematode infection using rats infected with Strongyloides venezuelensis, as well as to describe the consequences of a daily treatment with dexamethasone, which has been described as an efficient immunomodulator of the pulmonary alterations in the asthmatic process.

MATERIALS AND METHODS

Animals.

Specific pathogen-free (SPF) male Wistar rats weighing 100–120 g, bred at the Department of Parasitology of the Universidade Estadual de Campinas (UNICAMP), were used in the experiments. During this study, the rats were maintained at the animal facilities of the Faculdade de Ciências Farmacêuticas de Ribeirão Preto (University of São Paulo, Brazil), fed with laboratory chow, and given tap water to drink ad libitum. The experimental procedures used received prior approval from the local animal ethics committee.

Parasites.

Strongyloides venezuelensis, a nematode parasite that obligatorily migrates through the host lungs before establishment in the duodenal mucosa, was used in the experiments. The nematode was isolated from rodents Bolomys lasiuris and was maintained at the Department of Parasitology (UNICAMP), by serial passage in Wistar rats. S. venezuelensis infective filiform (L3) larvae were obtained from charcoal culture of infected rat feces, incubated at 28°C for 72 hours and collected and concentrated using the Rugai method.6 The recovered larvae were washed several times in phosphate-buffered saline (PBS) and counted. For each infection, 9,000 S. venezuelensis L3 larvae in 300 μL of PBS were inoculated in the abdominal region with a subcutaneous injection.

Experimental design.

Animals were divided into four groups: C, control group; CD, control and dexamethasone group; I, infected group; ID, infected and dexamethasone group. The infected groups (I and ID) were inoculated subcutaneously in the abdominal region with 9,000 S. venezuelensis infective larvae L3, and the controls (C and CD) were inoculated with 300 μL of PBS. The CD and ID groups received 2 mg/kg daily of dexamethasone disodium phosphate (Decadron; Aché Laboratories, Campinas, Brazil), initiated 1 hour before infection. At 1, 3, 5, 7, 14, and 21 days post-infection (dpi), six rats from each experimental group were killed.

Morphological analysis of the inflammatory process in the lungs.

The right lung was inflated with 10% buffered formalin using a tracheal cannula, and the left was immersed in this fixative for 48 hours. Sagittal sections of the lungs were obtained and processed for paraffin embedding. Histologic sections (5 μm in thickness) were cut and stained with hematoxilin and eosin (H&E).

Morphometric analysis of the inflammatory process in the lungs.

Morphometric analysis was performed with Leica QWin software (Leica Microsystems Image Solutions, Cambridge, UK) in conjunction with a Leica DMR microscope (Leica Microsystems, Wetzlar Germany), video camera (Leica Microsystems, Heebrugg, Switzerland), and an online computer. The analysis was evaluated by a researcher blinded to the protocol design (CTS). The eosinophils (H&E) and mast cells (Luna stain)7 present in the peribronchovascular tissue (axial septum) were counted in 10 randomly non-coincident fields, at a magnification of ×400. The total area was 0.8 mm2. Randomly selected areas were also used to evaluate the eosinophilic infiltration observed in the alveolar parenchyma. Ten fields containing well-developed granulomas in the I group and 10 fields of agglomerated inflammatory cells in the ID group were selected from the slices to be analyzed. The magnification was ×400, giving a total area of 0.8 mm2. The means were calculated and the values expressed as cells/area (mm2).

For evaluation of the reticular fibers in the alveolar parenchyma, the slides were stained with Gomori reticulin.8 The surface density of reticular fibers in the lung was determined by optical density in the images analysis. The system used consists of a video camera (Leica Microsystems) applied to a Leica microscope DMR (Leica Microsystems) and attached to a computer. The images were processed by Leica QWin software (Leica Microsystems Image Solutions). The thresholds for reticular fibers were established for each slide, after enhancing the contrast up to a point at which the fibers were easily identified as black bands. Bronchovascular bundles were carefully avoided during the measurements. Ten randomly selected inflammatory foci presented in the alveolar parenchyma were considered for each group, at ×400. The total area examined for group was 0.8 mm2. The controls groups were counted in the distal parenchyma, independent of inflammatory agglomerates. The means were calculated, and the values are expressed as percentage (%).

The macrophages were counted in the alveolar parenchyma using immunohistochemical slides. The sections were mounted on silane-coated slides, deparaffinized, washed in phosphate-buffered saline, and submitted to heat-induced antigen, endogenous peroxidase inhibition, and nonspecific antibody antigen block. Subsequently, the sections were incubated with the primary antibody CD68 (clone ED-1, 1/100; Serotec, London, UK). Stereologic analysis was performed to evaluate the spatial distribution of the macrophages in the alveolar parenchyma, using an integrating eyepiece with a coherent system made of a 100-point grid coupled to a conventional light microscope (BH2; Olympus, Tokyo, Japan). The number of points of the integrating piece falling on macrophages was counted in a total of 20 random non-coincident microscopic fields, at ×400. The total area examined was 0.12 mm2. The values were expressed as percentage (%).

Statistical analysis.

Data are expressed as the mean ± SEM. Differences between groups were tested by one-way analysis of variance (ANOVA) and the Newman-Keuls post-hoc test. For differences between two groups, the Student t test was used. A 5% level of significance was chosen to denote significant differences between the means. Results were analyzed using the GraphPad Prism 4.0 statistic program (GraphPad Software, San Diego, CA).

RESULTS

Animal growth.

There was no mortality during the experiment. The C and I groups experienced body weight gain (145.6 ± 10.1 and 178.6 ± 18.6 g, respectively), whereas all animals treated with dexamethasone (CD and ID) showed severe body weight loss: 102.2 ± 2.1 and 101.3 ± 0.5 g, respectively (P < 0.001). They appeared extremely slim, with an important reduction of the subcutaneous fat. No apparent respiratory insufficiency was observed at any time during the experimental period.

Morphological examination of the lungs (gross and histopathologic findings).

At 1 dpi, L3 larvae that had migrated through the pulmonary parenchyma were seen in the alveolar spaces. They produced scattered foci of alveolar hemorrhage, accompanied by an eosinophilic infiltrate in the peribronchovascular tissue (axial septum) and a diffuse increase in alveolar macrophages. Infected animals treated with dexamethasone yielded similar findings in the pulmonary parenchyma, although less prominent in all aspects, including the inflammatory infiltrate. At 3 dpi, multiple hemorrhagic areas could be seen in the pleural surface of infected animals. Microscopically, these corresponded to hemorrhagic foci with larvae between cuticle fragments, intra-alveolar proteinaceous exudates, and inflammatory cells, notably eosinophils in large numbers in the axial septum, accompanied by a less prominent increase in the intra-alveolar macrophages. The corresponding treated group showed more confluent hemorrhagic areas, increased larvae and cuticles, and a similar inflammatory infiltrate of eosinophils and macrophages, although less prominently. At 5 dpi, the inflammatory process was notably granulomatous in all infected animals. The eosinophilic infiltrate decreased in the axial septum, in this case being more evident in the alveolar parenchyma as a granuloma component. Eosinophils, giant cells, and macrophages were also present in the granulomas, which occasionally contained parasites. All components were surrounded by a loose network of reticular fibers. There were also more macrophages dispersed in the alveolar spaces, most commonly hemosiderin-laden and xantomatous macrophages. In contrast, no evident granuloma formations or parasites were visualized in the treated group. The eosinophilic infiltrate in the axial septum decreased relative to the third day. Hemosiderin-laden and xantomatous macrophages were more numerous, but distributed dispersedly in the alveolar parenchyma without any sign of organization. At 7 dpi, the granulomas were defined and compact, composed of macrophages and eosinophils that were entrapped in a well-developed and concentric reticular connective tissue network. Most of the macrophages were confined to the granulomas. Parasites were not visualized. The granulomas were seen grossly in the pleural surface. At the same time of the experiment, animals treated with dexamethasone continued to show no evidence of granulomatous inflammation. The macrophages appeared dispersed in the alveolar spaces, eventually forming loose agglomerates, but without any signs of organization. No reticular fiber networks were visualized entrapping inflammatory cells, and no eosinophils were distinguished in the distal parenchyma or in the axial septum. After 14 days of infection, the granulomas were scarcer in the alveolar parenchyma, showing a less compact appearance in the I group. The eosinophils practically disappeared in the axial septum but continued to be prominent in the granulomas, which were intermixed with macrophages, both entrapped in concentric arranged reticular fibers. No parasites were visualized. Treated animals in the ID group did not differ from the previous group, but free larvae could be seen in the alveolar parenchyma. There was no prominent alveolar hemorrhage seen, as in the initial days of the experiment, but hemosiderin-laden macrophages continued to be dispersed in the alveolar parenchyma. Ultimately, on day 21, only residual granulomas and a minimum inflammatory infiltrate were seen in infected animals. The infected-treated group showed no significant alterations compared with the 14th day. Eosinophilic infiltrate practically disappeared from the axial septum. No more larvae were noted in any slices examined, but hemosiderin-laden macrophages were still dispersed in the alveolar parenchyma. In controls groups C and CD, no evident morphological alterations could be seen in the pulmonary parenchyma. The most interesting aspects of these results are shown in Figure 1.

Mast cells and eosinophils in the axial septum.

The infected group showed a significant increase in mast cell infiltrate in the axial septum, relative to the ID and C groups on 3 (2.2 ± 0.5 cells/area) and 14 dpi (2.8 ± 0.5 cells/area; P < 0.001). Mast cells were reduced in the axial septum during all experiments in the CD group. Eosinophilic infiltration in the axial septum was confirmed in the infected animals, starting 24 hours after infection (8.8 ± 1.8 cells/area). It peaked on day D (67.5 ± 7.3 cells/area; P < 0.05) and decreased progressively until the end of the experiment, except on the last day of the experiment (20.7 ± 2.3 cells/area; P < 0.001). Infected-treated animals showed a less prominent eosinophilic infiltrate in the axial septum than the infected group but with a similar behavior during the experiment. The C and CD groups showed no significant eosinophil infiltration in the axial septum. The most interesting aspects of these results are shown in Figure 2.

Granuloma formation.

Almost no eosinophils were observed in the alveolar parenchyma before Day 5 of the experiment, when they were directly related to granuloma formation in the infected group (49 ± 1 cells/area on Day 7 and, 64.6 ± 16 cells/area on Day 14; P < 0.001, compared with the infected-treated group), nearly disappearing at the end of the experiment (3.6 ± 1.6 cells/area). None of the other groups showed relevant eosinophilic infiltration in the alveolar parenchyma. Macrophage density was progressively increased in the alveolar parenchyma until Day 5 (3 ± 0.2%; P < 0.001) in the infected group, when it began to decrease, returning almost to the basal values by the end of the experiment (1.2 ± 0.1%, 0.7 ± 0.1%, and 0.5 ± 0.1% at Days 7, 14, and 21, respectively). This coincided with the organization of the inflammatory infiltrate in granulomas initiated at that time. The ID group also showed an increased density of alveolar macrophages in lung parenchyma, but this increase was maintained throughout the experiment (0.6 ± 0.1%, 1.2 ± 0.1%, 2.3 ± 0.1%, 2.3 ± 0.1%, 2.0 ± 0.2%, and 1.9 ± 0.1%; P < 0.001). None of the other groups showed relevant macrophage infiltration in the alveolar parenchyma. Reticular fibers showed a conspicuous increase in the I group, directly related to the granuloma composition. These were prominent, entrapping parasites and inflammatory cells and exhibiting the usual geometric form of granulomas. They measured 6.2 ± 1.5%, 15.3 ± 1%, and 16.1 ± 2% on Days 5, 7, and 14, respectively, with statistical significance compared with the ID and control groups. At the end of the experiment, the reticular network tended to disappear together with the granulomas (5.0 ± 1.6%). The ID group also showed increased reticular fibers in the alveolar parenchyma, but these were visibly restricted to the alveolar septum. No reticular fibers were visualized interspersed with the loose agglomerates of macrophages in the lung parenchyma of the ID group. They measured 4 ± 0.3%, 3 ± 0.3%, and 6.8 ± 1% on Days 5, 7, and 14, respectively. Control groups showed no significant changes in the percentage of reticular fibers. The most interesting aspects of these results are shown in Figure 3.

DISCUSSION

To gain insight into the mechanisms underlying dexamethasone-induced alterations in the inflammatory responsiveness during the pulmonary cycle of S. venezuelensis, we infected Wistar rats with a substantial quantity of larvae and treated them with dexamethasone for 21days. Three principal effects were found: 1) increased alveolar hemorrhagic inflammation provoked by the passage of larvae into alveolar spaces; 2) significant decrease of eosinophil and mast cell migration to the axial septum of the lungs; and 3) impaired formation of the reticular fiber network, interfering with granuloma organization.

Increased alveolar hemorrhagic inflammation.

Infected rats showed a remarkably rapid resolution of mechanical damage and inflammation caused by S. venezuelensis larvae migrating through the pulmonary environment (Figure 1). The exact mechanism by which larvae break into the air spaces from the circulation is not known. However, considering that the average width of larvae (15–20 μm) is larger than the size of lung capillaries (7–10 μm), larvae in the circulation would embolize and break lung capillaries.9 Once inside the air spaces, larvae might be passively carried by ciliar movement of the bronchial epithelium, or they may actively crawl up the airway. They may sometimes be entrapped by a granulomatous process, which took place at Day 5 in the infected animals. However, both hemorrhage and granulomatous inflammation were largely resolved, and by 21 dpi, the histologic appearance of the pulmonary parenchyma and the large airways was nearly indistinguishable from that of uninfected controls (Figure 1). This rapid resolution was probably mediated by cytokines IL-4 and IL-13, which have been shown to mediate inflammation, epithelial cell hyperplasia, and mucus production in the lung in nematode infections as well as in the asthmatic process.10 In contrast, the larva-induced inflammation in the lungs of dexamethasone-treated animals persisted through 21 dpi, with significant levels of cellular infiltrate mainly consisting of hemosiderin-laden and xantomatous macrophages throughout the lung (Figure 1). The mechanistic basis for this difference in the ability to resolve pulmonary inflammation between these experimental groups is not clear, but it presumably reflects both the interference in the immunologic mechanisms and the increased presence of larvae passing through lung capillaries in treated animals. These alterations are reflected in the hemorrhagic aspect of the lung parenchyma, which appeared more prominent and confluent, with more larvae and many cuticles. In fact, studies have proposed that the administration of corticosteroids increases production of ecdysteroid-like molecules, hormones that control molting in S. venezuelensis larvae.11 Increased quantities of these substances may upregulate the molting rate, leading to an increased worm burden, accelerating the transformation of rabditiform to invasive filariform larvae, and promoting hyperinfection and dissemination to larvae in various organs.13 In this study, looking beyond the lungs, larvae were found in the liver, spleen, kidney, heart, and brain at 14 dpi in infected-treated animals accompanied by increased females number in the small intestine lumen (data not shown), confirming hyper-infection. The increased presence of larvae passing throughout the pulmonary capillaries and the altered inflammatory response may explain these findings. Infected rats are healthy and free of infection after 21 days, whereas the infected-treated animals showed extreme subnutrition, with an important fat subcutaneous depletion, indicating a poor prognostic.

Eosinophil and mast cell migration to the axial septum.

Eosinophils are end-stage granulocytes that derive from the bone marrow under the influence of IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF).14 Under baseline conditions, they leave the bone marrow and migrate to the gastrointestinal tract, where they reside within the lamina propria.15 In some inflammatory conditions such as nematode infections that have a pulmonary cycle, eosinophils migrate to extraintestinal tissues such as the lung, where they are thought to affect the airways in a number of ways.16 In vitro, toxic granule proteins such as major basic protein, eosinophilic cationic protein, and eosinophil peroxidases have a direct cytotoxic effect on the respiratory epithelium, enhance airway smooth muscle responsiveness, and trigger mast cell degranulation.17,18 Mast cells synthesize and secrete a number of pro-inflammatory cytokines including IL-4, IL-5, and IL-13, which regulate both IgE synthesis and the development of eosinophilic infiltration.19 Eosinophils also secrete a number of pro-inflammatory interleukins, including IL-2, IL-4, IL-5, IL-10, IL-12, and IL-13, which enhance the Th2 response, and the profibrotic cytokine transforming growth factor-β (TGF-β), which is involved in airway remodeling.20,21 In our study, infected-treated animals showed a significant reduction of mast cell and eosinophil infiltration in the peribronchovascular interstitium (axial septum) during the experiment (Figure 2), suggesting that dexamethasone treatment can act on this migration.22 Interestingly, most of the immune aspects of nematode infection show similarities with those observed in asthma, raising obvious questions about the relationship between helminth infections and allergic disorders. In fact, glucocorticoids have been shown to reduce the numbers of mast cells in airway mucosal biopsy specimens from human subjects with mild atopic asthma.23 Also, they inhibit recruitment of eosinophils to the sites of inflammation after allergen provocation in animal models.2426 Because airway eosinophilia has been linked to the activation of CD4+ T cells that release Th2 cell–derived cytokines such as IL-4, IL-5, and IL-13 and chemokines such as eotaxin, it is generally believed that steroids act predominantly by inhibiting the generation of these cytokines and chemokines.27

Granuloma formation.

We found a different pattern of granulomatous response between infected (I) and infected-treated (ID) animals. The I group showed organized granulomas composed of eosinophils, macrophages, and multi-giant cells involved in a delicate network of reticular fibers, mainly corresponding to collagen type III populations. The concentric arrangement of the reticular fibers may serve to entrap the parasite, promoting anchorage of inflammatory cells and facilitating its elimination.28 The use of stereology, which gave us a better spatial idea of the inflammatory process, allowed us to show that most of the macrophages are integrated in these networks. In contrast, even in high quantities, alveolar macrophages of infected-treated rats did not show evident signs of organization, only loose agglomerates. Even when activated and probably producing TGF-β, one of the principal cytokines involved in the remodeling process,29 macrophages alone did not lead to development of these concentric fiber reticular networks. On the other hand, a role for eosinophils in host defense against specifically tissue-invading helminths has been suggested,30 but less attention has been given to a possible role for eosinophils in repair and remodeling processes, despite the well-documented association of tissue eosinophilia and eosinophil degranulation with several fibrotic syndromes.31 In fact, eosinophils are a source of several molecules implicated in tissue remodeling processes.32 These include TGF-β,33 TGF-β1,34 fibroblast growth factor-2 (FGF-2),35 vascular endothelial growth factor (VEGF),36 matrix metalloprotease-9 (MMP-9),37 tissue inhibitor of metalloproteinase-1 (TIMP-1),38 IL-13,39 and IL-17. Because eosinophils were not visualized intermixing with alveolar macrophage agglomerations in the infected-treated group, we hypothesized that they may have a pivotal role in development of the reticular fiber network (Figure 3). Thus, this study provides strong evidence that there is a causal relation between eosinophils and granuloma arrangement. Taken together, these findings strongly support the hypothesis that eosinophils contribute, to a greater or lesser extent, to the remodeled pulmonary parenchyma in nematode infections, and that eosinophil-derived TGF-β1 could be central in facilitating matrix deposition in the lungs. Although is out of the scope of this study, we can suggest that dexamethasone treatment interferes with cytokines unleashing the immune response against S. venezuelensis infection, culminating in this inefficient response in rats.

Our observations are similar to that seem in human lungs infected with Strongyloides stercoralis. Usually, they present filariform larvae in the alveoli, eosinophilic infiltrate, and hemorrhage.40 In patients treated with corticosteroids, accelerated rate of autoinfection with dissemination of larvae throughout the pulmonary parenchyma, massive alveolar hemorrhage, and reduced eosinophils effects were described.4143 These pathologic similarities can provide important targets in further studies of pathogenetic mechanisms of strongyloidiasis.

In conclusion, this paper showed that the use of drugs with immunomodulatory actions, such as dexamethasone, in addition to interfering with morbidity from the pulmonary cycle of S. venezuelensis infection, may contribute to showing the mechanisms involved in its pathogenesis. The mechanisms of cytokine-regulated survival and apoptosis by granuloma sub-populations have profound implications in pathology and are currently the subject of study in our laboratory.

Figure 1.
Figure 1.

Gross and microscopic aspects of the lungs observed in animals infected with S. venezuelensis (left) and infected-treated with dexamethasone (right). On Day 1, scarce hemorrhagic foci containing larvae are seen in the alveolar spaces of infected animals (inset: larva). A similar aspect is observed in infected-treated animals (arrows). The hemorrhagic foci are more prominent on Day 3 in both groups, showing intra-alveolar proteinaceous exudates, inflammatory cells, larvae (arrow), and cuticle fragments. This aspect is more severe in the infected-treated group, with confluent hemorrhagic foci. Larvae can be seen inside the rectangle. On Day 5, most of the hemorrhage is now composed of hemosiderin-laden macrophages that give a tan appearance to the pulmonary surface. Note the granulomatous aspect of the inflammatory infiltrate in the alveolar parenchyma (arrow). These aspects were not observed in the infected-treated group. The inflammation is still hemorrhagic, with prominent exudates and cuticle fragments. However, no larvae or granulomas are observed at this day in this group. After 7 days, the granulomas are well developed, composed by macrophages and eosinophils, and can be seen in the pleural surface of infected animals. The infected-treated group shows macrophage agglomerates without signs of granulomatous organization (arrow). On Day 14, the granulomas are scarcer in the alveolar parenchyma in the infected group, whereas in the infected-treated group, free larvae are seen in the alveolar parenchyma intermixed with hemosiderin-laden macrophages without granuloma arrangement. On Day 21, only residual granulomas are seen in infected animals and, in the infected-treated group, dispersed hemosiderin-laden macrophages are seen in the alveolar parenchyma. (H&E; original magnification, ×200; inset, ×640). Scale bars = 4 mm and 50 μm. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 4; 10.4269/ajtmh.2008.79.571

Figure 2.
Figure 2.

Eosinophilic infiltrate and mast cells (not seen in this stain) in the axial septum in controls, S.v.-infected animals, and infected-treated with dexamethasone. The most prominent eosinophilic infiltrate can be observed on Day 3 in the infected group. This infiltrate diminishes through Days 5 and 7 of the experiment. The infected-treated group shows a behavior similar to the eosinophilic infiltrate in the axial septum, although less prominently. The controls show no eosinophilic infiltration of the peribronchovascular tissue. (HE; original magnification, ×400). A, Mast cell distribution in the axial septum in all groups. Note the increase in mast cells on Days 3 and 14 after infection in the infected group, whereas the infected-treated group shows no increase in mast cells. B, Eosinophil distribution in the axial septum in all groups. Data are expressed as mean ± SEM. *P < 0.001, #P < 0.01, †P < 0.05. Scale bars = 50 μm. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 4; 10.4269/ajtmh.2008.79.571

Figure 3.
Figure 3.

Granulomatous formation in the alveolar parenchyma in S.v.-infected animals (left) and infected-treated with dexamethasone (right). The panel shows granulomas on Days 5, 7, and 14 in the infected group, composed by eosinophils and macrophages entrapped in a well-developed reticular fiber network, arranged concentrically. Note the presence of a larva in the center of the granuloma (arrow) at the fifth day after infection. In contrast, no evident reticular fiber organization was seen between the agglomerated macrophages in infected-treated animals. (HE and Gomori reticulin stain; original magnification, ×400). A, Eosinophils counted in the granulomas or inflammatory agglomerates in infected-treated animals. B, Volume fraction (%) of macrophages in the alveolar parenchyma. C, Reticular fiber densitometry (%) in the granuloma or around agglomerated macrophages in infected-treated animals. Data are expressed as mean ± SEM. *P < 0.001, #P < 0.01, †P < 0.05. Scale bars = 50 μm. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 4; 10.4269/ajtmh.2008.79.571

*

Address correspondence to Simone G. Ramos, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, São Paulo, Brazil. E-mail: sgramos@fmrp.usp.br

Authors’ addresses: Cristiane Tefé-Silva, Elaine M. Floriano, and Simone G. Ramos, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14049-900, Ribeirão Preto, São Paulo, Brazil, Tel/Fax: 55-16-36023341, E-mails: cristefe@usp.br, floriano@fmrp.usp.br, and sgramos@fmrp.usp.br. Daniela I. Souza, E-mail: daniela_i_souza@bd.com. Marlene T. Ueta, Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, 6109, 13073-970 Campinas, São Paulo, Brazil, Tel/Fax: 55-19-35216301, E-mail: mtu@unicamp.br. Lúcia H. Faccioli, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil, Tel: 55-16-36024303, E-mail: faccioli@fcfrp.usp.br.

Acknowledgments: The authors thank Abel Dorigan Neto, Carlos A. Sorgi, João B. A. Oliveira, and Maria Elena Riul for excellent technical assistance and Julio C. Matos for photography assistance.

Financial support: C.T.S. received a scholarship of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. S.G.R. and L.H.F. are investigators of Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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