Original article

Toxoplasma gondii and mast cell interactions in vivo and in vitro:experimental infection approaches in Calomys callosus

(Rodentia, Cricetidae)

Gabriela Lícia S. Ferreira a,b, José Roberto Mineo a, Juliana Gonzaga Oliveira b,Eloisa Amália V. Ferro b, Maria Aparecida Souza a, Ana Alice D. Santos b,*

a Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará, 1720, Campus Umuarama,38400-902 Uberlândia, MG, Brazil

b Laboratory of Histology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará, 1720, Campus Umuarama,38400-902 Uberlândia, MG, Brazil

Received 8 August 2003; accepted 14 November 2003

Abstract

In the present work, we studied some qualitative and quantitative characteristics of mast cells located in the peritoneal cavity, submandibu-lar and dorsal lymph nodes and ileum of Calomys callosus experimentally infected by Toxoplasma gondii. In uninfected animals, the majorityof mast cells had similar ultra-structural characteristics, including several cytoplasmic granules with homogeneous and electron densecontents. However, after 1 h of infection, a significant influx of mast cells into peritoneal cavity was observed. The number of mast cells in thiscompartment decreased progressively in infected animals, and was significantly lower than the number of mast cells in control animals after48 h of infection. Mast cells from infected animals or from purified suspensions that were infected in vitro presented significant morphologicalmodifications, suggesting a degranulation process: cytoplasmic granules with electron dense content, fusion of the cytoplasmic granules,intracytoplasmic channels, cytoplasmic granules with flocculent material, plasma membrane rupture and granule contents in the extracellularenvironment. A remarkable increase in the influx of neutrophils toward the peritoneal cavity of the infected animals was observed after 12 h ofinfection. Moreover, this event occurred after the mast cell degranulation process took place. The relative increase in the number of mast cellsand neutrophils was also followed by an increase in the number of macrophages, but there was a significant decrease in lymphocyte influx.After 48 h of infection, the parasite had spread from the peritoneal cavity to all organs examined. Also, mast cells from these organs showedevident morphological alterations, indicating the presence of the degranulation process. These results suggest that mast cells are deeplyinvolved with the acute phase of the inflammatory response in this experimental model.

© 2003 Elsevier SAS. All rights reserved.

Keywords: Toxoplasma gondii; Mast cells; Host cell–parasite interactions; Calomys callosus; Experimental infection

1. Introduction

Toxoplasma gondii, an obligate intracellular coccidian, isan important opportunistic pathogen for humans and animalsand it infects a wide range of hosts. It is one of the mostabundant protozoan parasites in the world, since approxi-mately 25% of the global human population is chronicallyinfected by T. gondii. It is responsible for important publichealth problems [1] due to congenital infections that may

result in severe birth defects, or as a result of reactivation ofchronic infections, which can lead to the development ofencephalitis, a particular problem for immunosuppressedpersons [2]. T. gondii is well known as a potent type 1 cytok-ine inducer, and while these cytokines are required to surviveinfection, their overproduction may lead to pathology anddeath of the host [3]. The survival of both the host and theparasite is dependent on some of these tachyzoites becomingencysted as slowly dividing bradyzoites in brain and muscle,while the remaining tachyzoites are eliminated [4]. Themechanisms that control the tachyzoite replication in theinfected tissues involve both an innate acute inflammatoryresponse and an antigen-specific inflammatory response.

* Corresponding author.Tel.: +55-34-3218-2240; fax: +55-34-3218-2333.

E-mail address: parreira@ufu.br (A.A. D. Santos).

Microbes and Infection 6 (2004) 172–181

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© 2003 Elsevier SAS. All rights reserved.doi:10.1016/j.micinf.2003.11.007

Such mechanisms are not completely understood, eventhough it has become clear that early events occurring duringthe innate response shape acquired immunity [5].

Mast cells have been regarded as a powerful cell type, dueto their effector and regulatory functions [6]. They are re-leased into the circulation as morphologically unidentifiedprecursors, and after being recruited to different tissues, theycomplete their differentiation. The morphological, biochemi-cal and functional heterogeneity of mast cells is based on theconcept that they mature and differentiate after settling intospecific tissues with certain microenvironmental factors, un-der the influence of stem cell factor and other locally pro-duced cytokines, as previously described in eutherian and innon-eutherian mammals [7–9]. Mast cells are distributedthroughout the connective tissue, where they are often situ-ated adjacent to blood and lymphatic vessels and beneathepithelial surfaces that are exposed to environmental anti-gens. Mast cells can release a group of preformed and newlysynthesized mediators, which are heterogeneous in their po-tency and biological function. These substances include thespasmogenic and vasoactive mediators and a plethora ofproinflammatory molecules, such as cytokines and chemo-kines, which can contribute to acute- and late-phase inflam-matory responses [6]. These mediators promote infiltrationof other leukocytes, including granulocytes, into the inflam-matory loci. Although there are a number of in vitro and invivo studies reporting the importance of mast cells in thedefense against pathogens [10], few reports have demon-strated an interaction between mast cells and T. gondii, andthe role of this interaction is not completely understood[3,11,12].

Based on the function of mast cells as an effector andregulatory cell type against pathogens, and because so little isknown about the interaction between mast cells and T. gon-dii, in the present study, we decided to investigate somequantitative and qualitative characteristics of mast cells fromperitoneal cavity, submandibular and dorsal lymph nodes andileum of Calomys callosus (Rodentia, Cricetidae) in the earlystages of infection by T. gondii tachyzoites. C. callosus, awild mouse-like autochthonous rodent from South America,is a reservoir for various infectious agents [13]. This animalis highly susceptible to T. gondii infection, and it has beenused as an appropriate alternative model to study experimen-tal toxoplasmosis [12,14–17].

2. Materials and methods

2.1. Animals

C. callosus from Canabrava lineage was used throughoutthe study. It belongs to a resident colony housed at theLaboratory of Histology of the Federal University of Uber-lândia [15,17]. The animals were kept in a 12-h light/12-hdark cycle in a temperature-controlled room (25 °C) withfood and water ad libitum. All animals were males, approxi-

mately 12 weeks old, weighing about 34 g when they wereinfected.

2.2. Parasites

Tachyzoites from the RH strain of T. gondii were main-tained by serial passage in Swiss mice by standard procedure,as described previously [18,19]. The viable parasites werecounted in a Newbauer hemocytometer chamber in suspen-sions containing trypan blue.

2.3. In vivo experimental infection

A group of C. callosus (n = 29) was inoculated intraperi-toneally with parasite suspensions containing 105 tachyzoi-tes of T. gondii in 0.2 ml of phosphate-buffered saline (PBS).The animals were sacrificed after 1, 3, 6, 12, 24, 36 and 48 hof infection. As control, another group of C. callosus (n = 25)was inoculated with peritoneal exudates from Swiss miceinoculated 48 h earlier with PBS only. Each time, cells fromthe peritoneal cavity were collected from both C. callosusgroups for morphological analysis by light or transmissionelectron microscopy. Fragments from the ileum, subman-dibular and dorsal lymph nodes were also analyzed after 48 hof infection.

2.4. Kinetics of peritoneal mast cells

Peritoneal cells were obtained from both infected(n = 28 per group) and uninfected animals (n = 28 per group),washed in PBS and stained with toluidine blue (0.5% tolui-dine blue O in 1% sodium borate). The numbers of mastcells, as well as the numbers of lymphocytes, macrophagesand neutrophils, in both groups for each interval—1, 3, 6, 12,24, 36 and 48 h—were counted in a Newbauer hemocytom-eter chamber in at least quadruplicate samples. Mast cellswere differentiated from other cells by their morphology andthe presence of metachromatic cytoplasmic granules.

2.5. In vitro experimental infection

Mast cell suspensions derived from C. callosus peritonealcells (n = 10) were purified by using Percoll density gradient(Sigma Chem. Co.). Briefly, volumes of 9 ml of the gradientwere added to the peritoneal cell pellet for 10 min at roomtemperature. After centrifugation at 300 × g for 10 min, thecells from the bottom phase were washed twice in RPMImedium plus 5% newborn calf serum. This purification pro-cess yielded cell suspensions containing about 90% mastcells. These cells were grown in the presence of tachyzoites,and after 0, 30 or 60 min, they were fixed, embedded in agar,and processed for transmission electron microscopy. As con-trols, mast cells were kept without parasites and processed atthe same way for electron microscopy.As controls, mast cellswere kept without parasites. After interaction, these cellswere fixed, embedded in agar, and processed for transmissionelectron microscopy.

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2.6. Light microscopy

Peritoneal cells from infected and uninfected animalswere obtained and washed with PBS. These cells were fixedin Carnoy’s solution containing ethanol, chloroform and ace-tic acid (6/3/1, vol/vol/vol) for 24 h at 4 °C and embedded inglycol methacrylate (Tecknovit 7100, Kulser). Then, 3-µmserial sections of this material were made (leaving a 30-µmspace between each section), and the sections were stainedwith 0.5% toluidine blue O and 1% sodium borate.

2.7. Transmission electron microscopy

Cells from the peritoneal cavity and fragments from il-eum, submandibular and dorsal lymph nodes were immersedand fixed in modified Karnovsky’s fixative solution (2.5%glutaraldehyde–2% paraformaldehyde in 0.1 M cacodylatebuffer at pH 7.2) for 17 h at 4 °C. After fixation, the speci-mens were post-fixed in reduced osmium for 1 h at roomtemperature, dehydrated in graded series of ethanol and em-bedded in Epon 812. After staining with uranyl acetate andlead citrate, sections were examined with a Zeiss EM-109electron microscope.

2.8. Morphological analysis of peritoneal cells afterinfection

The morphological analysis of the peritoneal cavity cellswas performed using a high-power objective (100) by count-ing intact and degranulated mast cells in each section. Thisanalysis was done by two investigators. At least 100 micro-scopic fields, two fields per section, from both infected(n = 8 per group) and uninfected (n = 4 per group) animalswere randomly selected. In parallel with the counting of mastcells, the numbers of neutrophils that were present in theperitoneal cavity in each group were determined.

2.9. Statistical analysis

Cells were counted using at least 25 serial sections or cellsuspensions. In all cases, data are expressed as mean ± S.D.Statistical differences between groups were determined us-ing the unpaired t-test with Welch’s correction (GraphPadSoftware, v.3.0, San Diego, CA, USA). Differences wereconsidered statistically significant when P < 0.01.

3. Results

3.1. Kinetics of mast cells in the peritoneal cavity frominfected and uninfected animals and their phenotypicmodifications

To determine the number of mast cells from T. gondii-infected animals at various intervals after infection, perito-neal cells were obtained from 1 to 48 h post-infection. In-

fected animals presented a different pattern of qualitative andquantitative characteristics from that of uninfected animals.One hour post-infection, a significant influx of mast cellstoward the peritoneal cavity, fourfold higher than in thecontrol group (P < 0.001) (Fig. 1A), was observed. Thenumbers of these cells decreased afterwards, similarly tothose from uninfected animals from 3 to 36 h, and became10-fold lower than uninfected animals 48 h post-infection(P < 0.001) (Fig. 1B). Mast cells from the peritoneal cavity ofuninfected animals exhibited a spherical shape containingmetachromatic cytoplasmic granules. These granules filled

Fig. 1. Number of mast cells in the peritoneal cavity from 1 to 12 h (A) orfrom 1 to 48 h (B) post-infection: comparison with uninfected animals.C. callosus was inoculated intraperitoneally with 105 tachyzoites of T. gon-dii and killed after 1, 3, 6, 12, 24, 36 and 48 h. Control animals wereinoculated with peritoneal exudates from uninfected animals. The numbersof mast cells in both groups (n = 21 animals per group) for each interval werecounted in a Newbauer hemocytometer chamber in at least quadruplicatesamples. Mast cells were identified by their morphology and the presence ofmetachromatic cytoplasmic granules. Mean ± S.D. are given. *P < 0.001.

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almost all the cytoplasm and did not show signs of degranu-lation (Fig. 2A). Twelve hours post-infection, it was ob-served that peritoneal mast cells changed their size and shapeand exhibited lower numbers of granules, with fusion of theirmembranes and the formation of intracytoplasmic channels(Fig. 2B–D). Some of these mast cells exhibited evidence ofdegranulation and parasitophorous vacuoles containing vari-able numbers of tachyzoites (Fig. 2C,D). At this time, theanalyses of mast cells revealed that in uninfected animals, thenumber of non-degranulated cells was significantly higherthan the number of degranulated cells, whereas in infectedanimals, the number of non-degranulated mast cells wassignificantly lower than the number of degranulated cells(P < 0.001) (Fig. 3A). Regarding the number of mast cellsharboring parasites, this number was much lower than thenumber of cells without parasites (P < 0.001) (Fig. 3B).

Uninfected mast cells cultured in vitro exhibited no mor-phological alterations after the purification process, and a

great number of homogeneous cytoplasmic granules withelectron dense material was seen. At the beginning of theinteraction between mast cells and T. gondii, no alterationswere observed either in host cells or parasites. However, after30 and 60 min, the mast cells exhibited a decrease in theelectron density of the granules, but showed granule fusionand an increase in the amount of their cytoplasmic projectionin the contact area with the parasites (Fig. 4A–C). Freetachyzoites with morphological alterations, as well as para-sitophorous vacuoles inside mast cells, were found after30 or 60 min.

3.2. Evaluation of the neutrophil influx in peritoneal cellsamong infected and uninfected animals compared withother cell populations

When determining the influx of inflammatory cells intothe peritoneal cavity, in parallel with the influx of mast cells

Fig. 2. Morphology of mast cells from the peritoneal cavity of uninfected C. callosus (A) or after 12 h of infection with T. gondii (B–D). A: Non-degranulatedmast cells (→) with metachromatic cytoplasmic granules. Bar scale: 12 µm. B: A partially degranulated mast cell (→). Bar scale: 12 µm. C: A degranulated mastcell (→) showing parasitophorous vacuoles containing tachyzoites (arrowhead). Bar scale: 12 µm. D: Electron micrograph of a mast cell and a neuthrophil. Themast cell shows cytoplasmic granules with heterogeneous contents and the fusion of their membranes forming intracytoplasmic channels (*) and parasitopho-rous vacuoles containing tachyzoites (**). Neutrophil (N). Bar scale: 2 µm. Inserts: bar scale: 0.6 µm.

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in this compartment, the most noteworthy event observedwas the significant increase in the neutrophil influx towardthe peritoneal cavity, 12 h post-infection (Fig. 5A). Theneutrophil influx for infected animals was 30-fold higherthan that found for uninfected animals (P < 0.001). Thenumber of neutrophils harboring parasites was significantlylower than neutrophils without parasites (Fig. 5B)(P < 0.001).As observed in Table 1, the significant neutrophilinflux that occurred in the infected animals from 12 to 48 hpost-infection was concomitant with a relative increase in thenumber of macrophages, whereas a relevant decrease in thenumber of lymphocytes occurred in the same period.

3.3. Evaluation of phenotypical modifications in mast cellsfrom lymph nodes and ileum of infected animals

To determine ultra-structural modifications in mast cellsfrom organs that could provide evidence of the systemicburden of the parasite, tissues from uninfected and infectedanimals were analyzed comparatively, 48 h post-infection. Insubmandibular and dorsal lymph nodes from control ani-mals, mast cells exhibited numerous cytoplasmic granuleswith homogeneous and electron dense contents, which werefound in the connective tissue of the capsule, lymphatic sinusand lymphoid tissue (Fig. 6A–C). In experimental animals,tachyzoites were detected in various regions of the subman-dibular and dorsal lymph nodes, including the connectivetissue from capsules. Inside the capsules, lysed cells contain-ing tachyzoites were frequently observed (Fig. 6D). Ta-chyzoites were seen inside and outside of the parasito-phorous vacuoles in lymphoid tissue and lymphatic sinuscells. Mast cells exhibited cytoplasmic granules with homo-geneous and electron dense contents, but the majority ofthem presented cytoplasmic granules with flocculent mate-rial and electron lucid halo. In addition, they presented gran-ule fusions, outlining wide intracytoplasmic channels, prob-ably due to the degranulation process (Fig. 6E, F).

Fig. 3. Number of degranulated and non-degranulated mast cells in perito-neal cavity from uninfected or infected animals, 12 h post-infection. C. cal-losus was inoculated intraperitoneally with 105 tachyzoites of T. gondii andkilled after 12 h. Control animals were inoculated with peritoneal exudatesfrom uninfected animals. A: Comparison between infected (n = 8) anduninfected (n = 4) animals. B: Comparison between mast cells harboringparasites and mast cells without parasites in the group of infected animals.Glycol methacrylate-embedded cells were subjected to serial sections (3 µmeach, leaving 30 µm space between each section) and stained with 0.5%toluidine blue O and 1% sodium borate. At least 100 microscopic fields wererandomly selected, two fields per section. Mast cells were identified by theirmorphology and the presence of metachromatic cytoplasmic granules.Mean ± S.D. are given. *P < 0.001.

Fig. 4. Experiments in vitro. A: Mast cell after 30 min of incubation with T. gondii, interacting with a parasite (Í). Note the increase in the amount of cytoplasmicprojections of the mast cell in the contact area with the tachyzoite. Bar scale: 1.2 µm. B: Detail of interaction (→) between mast cell and tachyzoite showed inA. Bar scale: 0.3 µm. C: Mast cell after 60 min of incubation with T. gondii, showing cytoplasmic granule fusion (*); granules exhibiting flocculent content withmoderate electron density and electron lucid halo (Í); extrusion of the content of the granule (c) and large cytoplasmic projections. Bar scale: 0.7 µm.

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Tachyzoites were found among granule contents secreted bymast cells (Fig. 6E). Mast cells that showed morphologicalalterations were observed apparently interacting with otherparasitized cells, including macrophages (Fig. 6G and in-sert). Extracellular parasites appeared to be destroyed duringthe interaction with mast cells, which exhibited morphologi-cal modifications typical of a degranulation process(Fig. 6H).

Forty-eight hours post-infection, a similar pattern of mastcell degranulation in the ileum was observed (data notshown). Extracellular tachyzoites were observed surround-ing the ileum lumen (Fig. 7A) and in the ileum submucosa(Fig. 7B). At this time of infection, tachyzoites were alsoseen inside parasitophorous vacuoles (Fig. 7C) as were extra-cellular tachyzoites in the ileum serosa (Fig. 7D).

4. Discussion

In the present study, it was observed that C. callosus mastcells from uninfected animals present a morphologic homo-geneity in various tissues from the examined organs. Indeed,mast cells from this rodent have characteristics similar tothose found in mast cells from other mammals like the rat,dog, sheep, human [7] and the opossum [8,9]. However, mastcells from T. gondii-infected animals or from purified sus-pensions cultured in vitro present a different pattern of quali-tative and quantitative characteristics than those from unin-fected animals. After 1 h of infection, a significant influx ofmast cells toward the peritoneal cavity was observed. Thenumbers of these cells decreased afterwards, being similar tothose from uninfected animals from 3 to 36 h, and becamelower after 48 h of infection. On the other hand, a high rate ofdegranulated mast cells was observed after 12 and 48 h ofinfection, when compared with control animals. Interest-ingly, even non-infected mast cells exhibited a high rate ofthe degranulation process after 12 and 48 h of infection. Thisfinding observed in infected animals is probably due to theearly presence of excreted–secreted antigens from T. gondiiin the peritoneal cavity and later on in other tissues [20].Thus, these antigens may be able to trigger mast cell de-granulation in the acute phase of inflammatory response.

Mast cells play an important role as effector cells ininflammatory responses. Preformed granules, released about5 min after antigen contact, contain histamine, proteoglycansand neutral proteases in various compositions. Lipid-derivedmediators include the metabolites of arachidonic acid bycyclooxygenase and lipoxygenase, which are produced about5–30 min after antigen contact. Various cytokines (IL-1, -3,-4, -5, -6, -8, -10, -13, -16, TNF-a, and TGF-b) and chemo-kines (MIP-1-a, MCP-1) are synthesized and secreted bymast cells about an hour after the antigen contact [6], andthese mediators have complex and partly redundant functionsin acute- and late-phase inflammatory responses. Mast cellsalso play a regulatory role in the immune response, becauseof their ability to secrete various mediators and cytokines,and present influence on T cell and B cell functions. It hasalready been mentioned that mast cells are the major sourceof certain cytokines (e.g. IL-4, -10 and -13) that can signalnaive and memory T cells to preferentially differentiate intothe Th2 subset. In addition, mast cells can present antigen toT cells, and in humans, they can directly stimulate IgEsynthesis in B cells [6].

In the present investigation, a remarkable increase in theinflux of neutrophils toward the peritoneal cavity of theinfected animals was observed 12 h post-infection. More-over, this event occurs after the mast cell degranulationprocess took place. The relative increase in the number ofmast cells and neutrophils was also followed by an increasein the number of macrophages but a significant decrease inlymphocyte influx. Earlier studies suggested that mast cell-deficient (KitW/KitW-v) mice displayed a significant defec-tive ability to recruit PMNs to the peritoneal cavity during

Fig. 5. Number of neutrophils in peritoneal cavity from uninfected orinfected animals, 12 h post-infection. C. callosus was inoculated intraperi-toneally with 105 tachyzoites of T. gondii and sacrificed after 12 h. Controlanimals were inoculated with peritoneal exudates from uninfected animals.A: Comparison between infected (n = 8) and uninfected (n = 4) animals. B:Comparison between neutrophils harboring parasites and neutrophils wi-thout parasites in the group of infected animals. Glycol methacrylate-embedded cells were subjected to serial section (3 µm each, leaving 30 µmspace between each section) and stained with 0.5% toluidine blue O and 1%sodium borate. At least 100 microscopic fields were randomly selected, twofields per section. Neutrophils were identified by their morphological cha-racteristics. Mean ± S.D. are given. *P < 0.001.

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early infection by T. gondii, suggesting that these cells serveas a major effector cell type involved in neutrophil recruit-ment [3]. It was also shown that CXCR2 is required for earlyneutrophil recruitment, since a significant impairment ofneutrophil influx was observed in CXCR2-deficient mice [3].

In the present study, after 48 h of infection, the parasitespread from the C. callosus peritoneal cavity to all organsexamined: submandibular and dorsal lymph nodes and il-eum. Mast cells from these organs presented evident mor-phological alterations, indicating the presence of the de-granulation process. Similar events were also seen afterinteraction between mast cells and T. gondii tachyzoites[11,12]. In addition, 48 h after T. gondii infection, C. callo-sus mast cells presented similar modifications to those seenin eutherian mammals under the effect of compound 48/80, awell-known degranulation agent [21].

As the animals were infected intraperitoneally in thepresent investigation, it can be assumed that the parasitesinvaded the mesentery and subsequently adjacent organs,including the ileum, through cell layers of mesothelium, andsoon after, through smooth muscle cell layers of the connec-tive tissue and the epithelial cell layers, where some of themreached the intestinal lumen. In another study, after intraperi-toneal infection with the RH strain, tachyzoites infected theorgans adjacent to the peritoneal cavity and later on infectedthe blood vessels, from which the tachyzoites spread throughthe whole organism [22]. If parasite dissemination from theperitoneal cavity to other organs occurs via the bloodstream,peripheral blood neutrophils, as well as monocytes and den-dritic cells, would be excellent candidates for transportingtachyzoites to other host tissues [17,23–25]. However, takenas a whole, neutrophils play a critical, dual role in restrictingtachyzoite growth; they lyse extracellular tachyzoites, andwhen infected, they retard intracellular tachyzoite division.

Mast cells found in the ileum submucosa 48 h post-infection showed significant morphological alterations andcontained T. gondii with altered morphology, probably in

response to the discharge of the granule contents from mastcells on the parasites present in this compartment. Massivedestruction of various cell types from ileum of mice infectedby the VEG strain of T. gondii has already been described.After 48 h of infection, the majority of cells from laminapropria were lysed, and only a small number of intracellulartachyzoites were seen in the process of division [1]. Recentdata from the literature have pointed out that regulation of theinflammatory process resulting from T. gondii infection inthe ileum is dependent on the secretion of mediators andcytokines produced by a variety of cellular interactions, andinclude the intraepithelial lymphocytes. This cellular cross-talking process seems to be essential in gut homeostasis afterinfection with this parasite [26–28]. In this context, Eimeriainfections trigger a substantial increase in the number ofintestinal mast cells, with consequent liberation of mast cellprotease II (RMCPII). This protease causes permeability ofthe mucosa, allowing the translocation of the immune systemelements inside the lumen [29]. Our results showed that mastcells from this compartment should also be included in thiscellular cross talk.

The ability of T. gondii to infect every type of nucleatedcell in mammals is not totally understood relative to theproteins or to the host cell receptors that participate in thisrelationship [30]. The success in invading, and consequently,replicating inside the host cells explains why so many celltypes, including mast cells, were observed to be parasitizedand destroyed in the organs analyzed in the present study,such as ileum and lymph nodes. Several parasitized cellulartypes and tachyzoites were seen in all areas of the subman-dibular and dorsal lymph nodes of C. callosus. These resultsare similar to those findings observed in mice infected by theVEG strain of T. gondii that, 48 h post-infection, have greatamounts of parasites in the mesenteric lymph node [1]. Ourdata also showed that mast cells from lymph nodes wereobserved interacting with extracellular parasites and with

Table 1Relative numbers of lymphocytes, macrophages, mast cells and neutrophils from peritoneal cavity of C. callosus infected by T. gondii RH strain, from 1 to 48 hpost-infection, when compared with uninfected animals

Time after infection (h) Group of animals(N = 7)

% Mean (±S.D.)Lymphocytes Macrophages Mast cells Neutrophils

1 Infected 91.3 (±5.7) 3.6 (±3.7) 3.7 (±3.7) 1.3 (±2.2)Uninfected 93.3 (±1.2) 1.0 (±1.7) 4.8 (±1.7) 0.9 (±1.6)

3 Infected 90.3 (±3.7) 2.6 (±2.9) 5.2 (±1.1) * 2.0 (±2.0)Uninfected 93.5 (±1.4) 0.7 (±1.1) 3.6 (±2.0) 2.3 (±2.2)

6 Infected 92.8 (±0.8) 2.9 (±1.0) 3.1 (±0.4) 1.2 (±0.2)Uninfected 94.7 (±2.3) 1.6 (±0.5) 2.8 (±0.9) 0.9 (±1.5)

12 Infected 81.1 (±5.5) 7.0 (±1.9) 4.1 (±0.9) * 7.9 (±2.9) *Uninfected 97.5 (±0.6) 1.1 (±0.0) 1.1 (±0.0) 0.3 (±0.6)

24 Infected 63.2 (±7.5) 4.7 (±1.6) 1.1 (±0.3) 31.0 (±6.8) **Uninfected 97.1 (±0.9) 0.5 (±0.9) 1.7 (±0.1) 0.6 (±1.0)

36 Infected 57.5 (±12.5) 12.5 (±3.9) 7.4 (±1.3) * 22.6 (±7.3) **Uninfected 92.4 (±5.9) 3.7 (±4.4) 2.8 (±2.8) 1.1 (±1.8)

48 Infected 72.3 (±3.9) 5.7 (±3.7) 6.3 (±3.2) * 15.7 (±0.9) **Uninfected 93.8 (±2.9) 2.5 (±2.2) 2.6 (±2.3) 1.1 (±1.9)

* P < 0.05; **P < 0.01, when infected and uninfected animals were compared.

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parasitized cells, and these interactions resulted in significantmorphologic alterations in those host cells.

In conclusion, our data suggest that mast cells are animportant cell type during the inflammatory response againstT. gondii. Parasite antigens induce the release of chemical

mediators by different mast cell populations. These cellpopulations may express significant variations in number,phenotype and function, particularly during the events thatcontribute to the acute phase of the inflammatory responseagainst this parasite.

Fig. 6. Electron micrographs of the dorsal lymph node from control animals (A–C) and from animals infected with T. gondii (D–H), after 48 h of infection. A:Mast cell in the connective tissue from capsule, showing numerous cytoplasmic granules with homogeneous and electron dense content. Bar scale: 2.4 µm. B:Mast cell in the lymphatic sinus. Bar: 2.4 µm. C: Mast cell in the lymphoid tissue. Bar scale: 2 µm. Both B and C exhibit the same characteristics described inA. D: Cell from capsule showing morphological alterations and a tachyzoite inside this cell (*). Bar scale: 1.2 µm. E: Disrupted lymphatic sinus mast cell withextrusion of cytoplasmic granules exhibiting flocculent and moderate electron density, wide intracytoplasmic channels (star) and a tachyzoite (*) among granulecontents secreted by that mast cell. Bar scale: 2 µm. F: Mast cell in the lymphoid tissue exhibiting intracytoplasmic channels with flocculent content, suggestinga degranulation process (*). Bar scale: 0.4 µm. G: Interaction between mast cell and macrophages harboring a parasitophorous vacuole (*). Mast cell in thelymphatic sinus exhibiting cytoplasmic granules presenting homogeneous and electron dense content with evident perigranular membrane and granules withflocculent contents and electron lucid halo. Bar scale: 2 µm. Insert: detail of some cytoplasmic granules with flocculent content and electron lucid halo. Bar scale:0.4 µm. H: Tachyzoite (*) with morphological alterations among granule contents secreted by mast cell. Bar scale: 0.4 µm.

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Acknowledgements

We thank Dr. Neide Maria Silva and Dr. Adriano MotaLoyola for critical reading of the manuscript, Hélgio Hein-isch Werneck for his technical assistance and Luiza SilvaPatriarca Mineo for review of the English. This work wassupported financially by Brazilian Research Agencies(CNPq, FAPEMIG and CAPES).

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