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Published online before print May 10, 2004
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Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
1Correspondence: Department of Veterinary Microbiology, WCVM, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4, Canada. E-mail: tabel{at}sask.usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: trypanosomes • liver • macrophages • immunity • cytokines • immunohistochemistry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The purpose of this study was to further investigate the effects of phagocytosis of T. congolense on Kupffer cells as well as the potential role of the IgM versus IgG class of antibodies specific for VSG. We used immunohistochemical double-label techniques to analyze the phagocytosis of T. congolense in vitro and in vivo. We found that IgM as well as IgG2a monoclonal antibodies (mAb) specific for the VSG mediated phagocytosis of T. congolense by macrophages [bone marrow-derived macrophage cell line from BALB/c (BALB.BM)] in vitro. It is more important that we provide direct evidence that phagocytosis of trypanosomes by Kupffer cells in vivo was mediated by passively administered IgM as well as IgG antibodies specific for the VSG. The concentrations of monokines, including interleukin (IL)-1ß, IL-10, and IL-12p40, were significantly increased in the plasma 6–48 h after phagocytosis. Phagocytosis of trypanosomes by Kupffer cells, mediated by actively synthesized antibodies, was first detected on day 5 and was dramatically enhanced on day 6 post-infection. In BALB/c mice infected for 6 days, treatment with IgM or IgG2a mAb specific for T. congolense VSG led to clearance of variant antigenic type (VAT) TC13 parasitemia but did not prevent death at the second parasitemia of a different VAT.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Parasite
T. congolense, Trans Mara strain, VAT TC13, was used in this study. The origin of this parasite strain has been described previously [18
]. Frozen stabilates of parasites were used for infecting CD1 mice immunosuppressed with cyclophosphamide, and passages were made every third day as described previously [18
]. The parasites purified from the blood of infected CD1 mice by diethylaminoethyl-cellulose chromatography [19
] were used for infecting BALB/c mice.
Antibodies
The rat hybridoma MCAP497 (specific for mouse macrophage antigen F4/80) was purchased from American Type Culture Collection (Manassas, VA). Biotin-conjugated goat anti-rat IgG was purchased from Cedarlane (Hornby, Ontario, Canada). Biotin-conjugated goat anti-rabbit IgG was purchased from Vector Laboratories (Burlingame, CA). Goat anti-rabbit IgG Alexa Fluor 546 and streptavidin Alexa Fluor 488 were purchased from Molecular Probes (Eugene, OR). Purified anti-mouse CD16/CD32 [Fc receptor for IgG III/II (Fc
III/IIR), clone 2.4G2] for blocking FcRs, recombinant mouse IL-1ß, IL-10, IL-12p40, and paired antibodies against mouse IL-1ß, IL-10, and IL-12p40 were purchased from PharMingen (San Diego, CA). We produced a polyclonal antiserum against T. congolense in rabbits [16
]. The production of mAb 6C1 (IgM), 1D11 (IgG2a), and 2D8 (IgG3), specific for the VSG of VAT TC13, has been described [20
].
Phagocytosis of trypanosomes in vitro and in vivo
To test phagocytosis of T. congolense in vitro, the retrovirus-immortalized BALB.BM cell lines of mice were used [21
]. BALB.BM (2x105) cells were grown in 0.4 ml complete medium, Dulbecco’s modified Eagle’s medium (DMEM)-10 [DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 mM 2-mercaptoethanol, and 100 IU penicillin and streptomycin (Life Technologies, Grand Island, NY)]. Cultures were performed in the Laboratory-Tek chamber slide system with eight wells (Nalge Nunc International, Napperville, IL) overnight at 37°C in a 5% CO2 atmosphere. Phagocytosis of trypanosomes was performed in the presence of mAb 6C1 (IgM) or 1D11 (IgG2a) at a concentration of 5 µg/ml with a constant macrophage: trypanosome ratio of 1:10. To allow for phagocytosis of trypanosomes, the chambers were incubated at 37°C for 1 h. Then, the chambers were rinsed with phosphate-buffered saline (PBS), the plastic case removed, and the cells on the microscope slide stained with immunofluorescent antibodies. To investigate antibody-mediated phagocytosis in vivo, BALB/c mice were injected with 3 x 108 VAT TC13 intravenously (i.v.). Thirty minutes later, the mice were injected with 1 mg mAb 6C1 (IgM) or 1D11 (IgG2a) in 200 µl PBS i.v. or 200 µl PBS alone as control. The mice were killed 1 h after the injection of antibodies. Liver sections were made for immunohistochemical stainings. To investigate the dynamics of phagocytosis, mediated by actively synthesized antibodies in vivo, BALB/c mice were infected with 103 VAT TC13 intraperitoneally (i.p.) and killed on days 0–7 post-infection. Liver tissues were collected for immunohistochemical staining for trypanosomal antigens and Kupffer cells.
Immunohistochemistry
For immunofluorescent double-staining of phagocytosis in vitro, BALB.BM cells cultured with trypanosomes in chamber slides were rinsed with PBS. FcRs were blocked with purified anti-mouse CD16/CD32 (FcIII/IIR) and 2% bovine serum albumin (BSA). Then, the slides were incubated with rat anti-mouse F4/80 mAb for 30 min. After three washes in PBS, the slides were incubated with biotin-conjugated goat anti-rat IgG. After three washes with PBS, the slides were incubated with streptavidin Alexa Fluor 488 (Molecular Probes) for 30 min, rinsed with PBS, and fixed with 5% formalin for 10–15 min. The slides were incubated with rabbit polyclonal antiserum against T. congolense in PBS containing 0.1% saponin for 30 min, rinsed with PBS, and followed by staining with goat anti-rabbit IgG Alexa Fluor 546 for 30 min. Saponin treatment is required for detecting intracellular antigens. As a control for demonstration of intracellular antigen, we also did immunofluorescent double-staining for macrophage antigens and trypanosomes on cells that had not been treated with saponin. Without the use of saponin, parasite antigen within the cytoplasma of BALB.BM cells was not detectable.
For immunoperoxidase staining of parasite antigen or Kupffer cells in liver sections, the liver tissues were deparaffinized in xylene and rehydrated through a graded series of ethanol. The tissues were incubated with proteinase K (EC 3.4.21.64, Sigma Chemical Co., St. Louis, MO; 20 µg/ml in 10 mM Tris/HCl, pH 7.4–8.0) for 30 min at 37°C. The tissues were rinsed and incubated with 3% H2O2 in methanol for 10 min, followed by blocking with 2% BSA in PBS. Then, the tissues were incubated with rabbit polyclonal antiserum against T. congolense for 30 min, rinsed in PBS, incubated with biotin goat anti-rabbit IgG, and followed by staining with horseradish peroxidase (HRP) streptavidin (Vector Laboratories). For staining of Kupffer cells, the tissues were incubated with culture supernatant fluids of rat hybridoma MCAP497 for 30 min, rinsed in PBS, incubated with biotin goat anti-rat IgG, rinsed in PBS, and followed by staining with HRP streptavidin. The tissues were rinsed and incubated with 3,3'-diaminobenzidine (substrate kit, Vector Laboratories) for color development. Finally, the sections were counterstained with hematoxylin, dehydrated, and covered with a coverslip. For immunofluorescent double-staining of the liver sections, the tissues were deparaffinized, treated with proteinase, and blocked with 3% H2O2 and 2% BSA as described above. The tissues were incubated with culture supernatant fluids of rat hybridoma MCAP497 for 30 min, rinsed in PBS, incubated with biotin goat anti-rat IgG, rinsed in PBS, and followed by staining with streptavidin Alexa Fluor 488. After three washes with PBS, the tissues were incubated with rabbit polyclonal antiserum against T. congolense for 30 min, rinsed in PBS, and incubated with goat anti-rabbit IgG Alexa Fluor 546 for 30 min.
Cytokine assays
Groups of four BALB/c mice were injected i.v. with 3 x 108 VAT TC13, followed by injection of 1 mg mAb 6C1 (IgM) or mAb 1D11 (IgG2a) in 200 µl PBS or 200 µl PBS alone 30 min later. The mice were killed 6, 24, or 48 h after injection of antibodies or PBS. Plasma samples were collected from mice and stored at –20°C for cytokine measurement. Plasma samples were also collected from BALB/c mice 5 and 6 days after infection with 103 VAT TC13. The levels of IL-1ß, IL-10, IL-12p40, or interferon- (IFN-) in plasma were determined by routine sandwich enzyme-linked immunosorbent assay (ELISA) using Immulon-4 plates (Dynax Technologies Inc., Chantilly, VA), according to the manufacturer’s suggested protocols (PharMingen). Each sample of plasma was tested for each cytokine in triplicate.
Measurement of sizes of Kupffer cells
Images of liver sections were taken by a digital camera, saved in the computer, and analyzed by a software of Northern Eclipse, version 6.0 (Empix Imaging Inc., Mississauga, Ontario, Canada). Each group included four individual animals, and one image was taken from each individual animal. The size of each image was chosen to be 90,500 square µ. The sizes of Kupffer cells were measured by using software of Northern Eclipse.
Estimation of parasitemia and determination of antigenic variant type and survival time
A drop of blood was taken from the tail vein of each infected mouse. The parasitemia was estimated by counting the number of parasites present in at least 10 fields at x400 magnification by phase-contrast microscopy. The presence or absence of VAT TC13 was determined by a motility test using mAb 2D8 as described previously [20
]. Briefly, 2 µl mAb 2D8 was mixed with 20 µl infected blood and incubated on ice for 10 min. All VAT TC13 trypanosomes become completely immobile within minutes, and only T. congolense of a VAT other than TC13 will remain motile. This is a rapid and convenient method to test for the presence of VAT TC13.
The survival time was defined as the mean number of days post-infection that the infected mice remained alive.
Statistical analysis
Data are represented as means ± SE. Significance of differences was determined by Student’s t-test or ANOVA using StatView SE 1988 software (Abacus Concepts, Berkeley, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Significant differences were also observed in the size of Kupffer cells after phagocytosis in vivo mediated by IgM or IgG2a compared with controls. However, there was no significant difference in the size of BALB.BM cells or Kupffer cells between IgM- or IgG2a-mediated phagocytosis (data not shown).
Dynamics of phagocytosis of trypanosomes by Kupffer cells of mice infected with T. congolense
To detect the dynamics of phagocytosis of parasites by Kupffer cells during infection, BALB/c mice were infected i.p. with 103 T. congolense VAT TC13. The infected mice were killed on days 0–7 post-infection. Immunoperoxidase staining showed that parasite antigens were undetectable in liver tissues from days 0 to 4 post-infection (Fig. 2A
). Parasite antigen was first detected in few Kupffer cells on day 5 post-infection (Fig. 2B)
. Markedly increased accumulation of parasite antigens in Kupffer cells was observed on day 6 post-infection (Fig. 2C) .
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in infected BALB/c mice. We found that the levels of IFN- in the plasma were enhanced three- to fourfold from day 5 (88.9±5.0 pg/ml) to day 6 (407.3±59.9 pg/ml). Thus, the dramatic increase of sizes of Kupffer cells from day 5 to day 6 correlated with analogous increases of IFN- levels in the plasma.
Plasma levels of monokines were significantly increased after phagocytosis of trypanosomes
We were wondering whether cytokines were secreted after phagocytosis of parasites. For this reason, we measured the plasma levels of some monokines such as IL-1ß, IL-10, and IL-12p40. BALB/c mice were infected with 3 x 108 T. congolense i.v. They were injected with 1 mg IgM or IgG2a mAb or PBS 30 min later. IL-1ß was undetectable in the plasma of all mice at 24 or 48 h post-injection of antibodies or PBS. We then measured the plasma levels of IL-1ß at 6 h post-injection of mAb or PBS alone, as it had been reported that secretion of IL-1ß by murine macrophages in vitro started 6 h after exposure of the macrophages to VSG of T. brucei rhodesiense [22
]. When compared with uninfected mice, we did detect a significant increase of IL-1ß in the plasma of infected mice treated with IgM (P<0.01) or IgG (P<0.05) at 6 h post-injection of mAb (Fig. 3A
). Plasma levels of IL-10 of mice treated with IgM (P<0.05 for 24 h; P<0.01 for 48 h) or IgG2a (P<0.01 for 24 and 48 h) significantly increased at 24 and 48 h post-injection of the mAb compared with normal, uninfected mice (Fig. 3B)
. IL-12p40 levels of mice treated with IgM (P<0.01) or IgG2a (P<0.01) mAb were also significantly enhanced at 48 h post-injection of the mAb, although there was no significant difference between mAb-treated mice and uninfected controls at 24 h post-infection (Fig. 3C)
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Treatment of the infected mice with IgM or IgG2a mAb specific for VAT TC13 led to clearance of TC13 parasitemia and increased survival but did not prevent death associated with the parasitemia of a different VAT
Finally, we treated mice, which had been infected with 103 T. congolense, with IgM or IgG2a mAb specific for VSG of VAT TC13 on day 6 post-infection. At this time, the mice had a parasitemia of 5 x 107 trypanosomes/ml blood. Initially, we treated the infected mice with 1 mg IgM or IgG2a mAb, as 1 mg mAb was enough to clear 3 x 108 TC13 when given 30 min after infection (see above). However, parasites could still be detected in the blood after injection of 1 mg mAb (data not shown). Then, another group of infected mice was injected with a total of 4 mg mAb. As shown in Figure 4
, the parasitemia was cleared on days 7 and 8 after treatment with IgM or IgG2a mAb on day 6. There was no significant difference between the second parasitemias of mice treated with 4 mg IgM or IgG2a mAb (Fig. 4)
. The untreated mice infected with T. congolense succumbed to their infections within 9–11 days with high parasitemia (Figs. 4
and 5
), of which 99% were of VAT TC13 (Fig. 4)
. In contrast, 50% of the infected mice treated with mAb survived for more than 17 days after infection (Fig. 5)
. The second parasitemias of the antibody-treated mice were 100% of a VAT (VAT TCx) different from VAT TC13. The survival of infected mice treated with IgM (P<0.01) or IgG2a (P<0.05) mAb was significantly increased as compared with untreated controls (Fig. 5)
. However, there was no significant difference in survival between IgM- and IgG2a-treated mice (Fig. 5)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous results demonstrated that tremendous amounts of trypanosomal antigens were located in Kupffer cells of susceptible BALB/c mice infected with T. congolense [16
]. However, the mechanism of phagocytosis remains to be determined. In this study, we found that significant amounts of parasite antigen accumulated in some cells of the liver of BALB/c mice, which were infected with 3 x 108 T. congolense VAT TC13 and 30 min later, received IgG2a or IgM mAb specific for VSG of VAT TC13. No phagocytosed antigen was found in infected mice, which received PBS only. Immunofluorescent double-staining further demonstrated that the antigen-containing cells were Kupffer cells (Fig. 1H and 1I)
. The accumulation of trypanosomal antigen in Kupffer cells was associated with failure to detect parasitemia. Thus, we have provided the first direct evidence that IgG2a as well as IgM anti-VSG antibodies mediated phagocytosis of trypanosomes by Kupffer cells in vivo. Macrophages have FcRs. It is possible that phagocytosis of trypanosomes mediated by IgG2a mAb is dependent on FcRs [26
]. The mechanisms of phagocytosis of African trypanosomes, mediated by IgM anti-VSG, are presently unknown. Although a FcR (Fc
/µR) for IgM has been described to be expressed on macrophages [27
], the biological significance of this Fc/µR has still to be defined. It has been shown that IgM antibodies specific for VAT TC13 activated complement and deposited murine C3b onto T. congolense [28
]. We recently found that complement receptor CR3 is one of the receptors involved in IgM antibody-mediated phagocytosis of T. congolense by peritoneal macrophages (Pan et al., unpublished).
One of the outcomes of phagocytosis was up-regulation of production of monokines such as IL-1ß, IL-10, and IL-12p40 in our model. IL-1ß was secreted within the first several hours post-infection. This finding is in the line with previous data from in vitro studies [22
]. We have no direct evidence to indicate that Kupffer cells secreted these cytokines. However, it is unlikely that antigen-specific lymphocytes secreted the cytokines within 6–48 h after infection. We suggest that splenic macrophages and/or Kupffer cells contributed to the enhanced synthesis of these cytokines. It is interesting that monokine levels in plasma of T. congolense-infected mice were enhanced even without injection of antibodies. In other words, monokines were secreted even without apparent phagocytosis of parasites, as we found that parasites were located outside of Kupffer cells in the absence of antibodies (Fig. 1D
and 1G)
. One possible explanation for this observation is that the preparation of 3 x 108-purified parasites used for inoculation might have contained a certain number of damaged or dead trypanosomes, which were phagocytosed and were sufficient to induce cytokine production in macrophages. The detection of their phagocytosis might have been below the sensitivity of the immunohistochemistry technique. In addition, it has been found that African trypanosomes shed VSG into the plasma of infected mice [29
30
31
]. Soluble VSG (sVSG) can bind to and stimulate macrophages to synthesize cytokines [22
, 31
32
33
]. It is surprising that infected mice treated with PBS produced significantly more IL-12p40, particularly at 48 h (Fig. 3C)
. At this time, the PBS-treated mice had a very high parasitemia, 
109/ml. The continual shedding of VSG from such a high parasitemia might have yielded great amounts of sVSG, which in turn, might have stimulated Kupffer cells and splenic macrophages to produce the high levels of IL-12p40. In fact, the story might be even more complex. The process of antibody-mediated phagocytosis of trypanosomes, although eliciting macrophages to produce more IL-12p40 than being produced by resting macrophages (Fig. 3C)
, might for yet unknown reasons, elicit a weaker signal for inducing synthesis of IL-12p40 than the binding of sVSG to macrophages by itself.
Phagocytosis of trypanosomes by Kupffer cells in BALB/c mice infected with 103 VAT TC13 could first be detected on day 5 (Fig. 2B)
. A dramatic increase of phagocytosis occurred on day 6 post-infection (Fig. 2C)
. In correlation, the Kupffer cells were significantly enlarged on day 6 post-infection, indicating a high state of activation of Kupffer cells (Fig. 2F)
. The dynamics of phagocytosis and Kupffer cell activation was correlated with at least three parameters: rising parasitemia (Fig. 2)
, increasing plasma levels of detectable IgM anti-VSG [17
], and marked increases of plasma levels of IFN- from day 5 to day 6 (see Results; ref. [34
]). There is a puzzling, still unanswered question: Why do the susceptible BALB/c mice fail to control the parasitemia in spite of demonstrable IgM anti-VSG antibodies appearing earlier in infected BALB/c than in infected C57BL/6 mice [17
] and the presence of the highly activated Kupffer cells (Fig. 2C
and 2F)
?
With the use of radioactive techniques, antibody-mediated clearance of 75Se-labeled trypanosomes in mice infected with T. brucei has been reported [14
, 15
]. The majority of labeled parasites was concentrated in the liver. Less were present in spleen and blood [14
, 15
]. In this study, we first infected BALB/c mice with 103 T. congolense and then treated the infected mice with IgG2a or IgM mAb specific for TC13 VSG on day 6 post-infection. IgG2a or IgM mAb (1 mg) was not sufficient to clear the parasitemia of 5 x 107 parasites/ml in BALB/c mice infected for 6 days. However, 1 mg mAb cleared the parasitemia of 108 parasites/ml, when the mAb were injected 30 min after infection. What made the difference? During the 6 days of infection, the parasites presumably shed a lot of sVSG [29
, 31
]. This process might interfere with phagocytosis of trypanosomes at two levels. First, the shed sVSG might bind a lot of anti-VSG antibody and thus is not available for the whole trypanosome. Second, free sVSG and/or complexes of the sVSG/anti-VSG antibodies might be phagocytosed by the Kupffer cells and thus mediate internalization of crucial receptors, which are not available at optimal numbers for the whole trypanosomes.
The parasitemia of infected mice was cleared by administration of the large amounts of 4 mg mAb (Fig. 4A) . The survival of infected mice treated with the mAb significantly increased as compared with untreated control. However, the treatments did not prevent death associated with a second parasitemia of a variant other than TC13. There was no significant difference between the effects of treatments with IgG2a or IgM mAb regarding parasitemia (Fig. 4A) and survival time (Fig. 5) of infected mice. This seems to suggest that IgG2a and IgM, in terms of protein concentrations, are equally efficient in mediating phagocytosis.
In summary, our results clearly indicated that IgM and IgG2a mAb specific for VSG could mediate phagocytosis of trypanosomes by macrophages in vitro and by Kupffer cells in vivo, and phagocytosis of trypanosomes was associated with profound changes of Kupffer cells.
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ACKNOWLEDGEMENTS |
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Received October 22, 2003; revised February 9, 2004; accepted April 13, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mediates early mortality Eur. J. Immunol. 33,108-118[CrossRef][Medline]
after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide J. Immunol. 145,3450-3455[Abstract]
/µ receptor mediates endocytosis of IgM-coated microbes Nat. Immunol. 1,441-446[CrossRef][Medline]
-induced nitric oxide production via reduction in STAT1 phosphorylation in African trypanosomiasis J. Immunol. 171,1466-1472 antibodies alters trypanosome-susceptible mice to a resistant-like phenotype J. Immunol. 161,5507-5515This article has been cited by other articles:
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M. Shi, G. Wei, W. Pan, and H. Tabel Experimental African Trypanosomiasis: A Subset of Pathogenic, IFN-{gamma}-Producing, MHC Class II-Restricted CD4+ T Cells Mediates Early Mortality in Highly Susceptible Mice J. Immunol., February 1, 2006; 176(3): 1724 - 1732. [Abstract] [Full Text] [PDF]
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M. Shi, G. Wei, W. Pan, and H. Tabel Impaired Kupffer Cells in Highly Susceptible Mice Infected with Trypanosoma congolense Infect. Immun., December 1, 2005; 73(12): 8393 - 8396. [Abstract] [Full Text] [PDF]
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