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International Immunology, Vol. 11, No. 3, 341-349, March 1999
© 1999 Japanese Society for Immunology

Differential CD86/B7-2 expression and cytokine secretion induced by Toxoplasma gondii in macrophages from resistant or susceptible BALB H-2 congenic mice

Hans-Georg Fischer, Ruth Dörfler, Bartholomäus Schade and Ulrich Hadding

Institute for Medical Microbiology and Virology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany

Correspondence to: H.-G. Fischer


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The influence of the intracellular parasite Toxoplasma gondii on macrophage expression of co-stimulatory molecules was studied. Unlike surface expression of CD80/B7-1, that of CD86/B7-2 is increased in mouse peritoneal macrophages 24 h following exposure to live toxoplasma in vitro. Most CD86 molecules are found on infected cells bearing a maximum parasite load. Consistent with the elevated membrane expression, the quantity of CD86 gene transcript is increased in macrophages infected by T. gondii in vitro or in vivo. CD86 up-regulation contributes to the augmented capacity of parasitized macrophages to present antigen to tuberculin-specific CD4+ T cells as demonstrated by blocking CD86 ligand interaction. T. gondii triggers up-regulation of CD86 in macrophages from BALB/c mice which are resistant to the development of toxoplasmic encephalitis. Infection of macrophages from the susceptible strain BALB.B, however, results in a decreased surface expression of CD86, although the parasite load and intracellular proliferation proved comparable in both macrophages. This differential host cell reaction correlates with disparate profiles in T. gondii-induced cytokine secretion. Upon challenge with toxoplasma, IL-1{alpha} and tumor necrosis factor (TNF)-{alpha} are released to a significantly higher extent by BALB/c than by BALB.B macrophages, whereas the latter secrete more IL-12 and IL-10. In BALB.B macrophages, T. gondii-induced IL-10 down-regulates surface expression of CD86, thus indicating an interference of parasite-dependent cytokine release and modulation of CD86. The biased secretory response in macrophages from the two congenic strains implies an MHC-dependent and dichotomous monokine induction by T. gondii. Up-regulation of CD86 seems to occur along the IL-1/TNF-inducing pathway and experimental evidence indicates that this enhances T cell activation by parasitized macrophages.

Keywords: CD80/B7-1, host cell reaction, IL-1{alpha}, IL-10, IL-12, monokine profiles, tumor necrosis factor-{alpha}


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The lymphocyte activation antigens of the B7 family, CD80 (B7-1) and CD86 (B7-2), are genetically and structurally related membrane-bound molecules (1–3) expressed on antigen-presenting cells (APC). Both are ligands for CD28 on the T cell surface and can deliver the co-stimulatory signal 2 required for TCR-dependent activation of resting T cells upon interaction with APC (reviewed in 4). Engagement of CD28 prevents T cell unresponsiveness. Furthermore, CD80 and CD86 can bind to the T cell activation antigen CTLA-4 (5) which mediates down-regulatory or co-stimulatory signals (6,7). It is yet unclear whether CD80 and CD86 bind to identical sites of their ligands as implied by the induction of similar co-stimulatory effects (8). However, recent evidence indicates functional differences between the two molecules (9,10). Both CD80 and CD86 have been detected on monocyte/macrophages, dendritic cells, and B and T lymphocytes (11), their expression is regulated by cytokines as well as by contact with infectious agents or their constituents (12).

Cell-mediated immunity is crucial in host defense against Toxoplasma gondii, an intracellular protozoan parasite frequently causing life-threatening disease in immuno-compromised individuals (13). In the mouse model, protective immunity requires the cooperation of macrophages, NK cells and T cells, and IFN-{gamma} is the pivotal mediator inducing anti-toxoplasma effector mechanisms. Susceptibility to toxoplasmosis depends on the host genotype (reviewed in 14), one resistance locus involved being linked to the MHC (15). As a T cell-independent pathway of host resistance and presumably of immunopathogenesis, T. gondii can directly stimulate macrophages, spleen dendritic and brain glial cells to express and secrete cytokines (16–19). This acute host cell reaction is triggered by so far unidentified parasite factor(s) and has proven to be distinct from the cytokine response to bacterial lipopolysaccharide (17,19). In the present study, we analyze T. gondii-induced activation of macrophages with respect to their expression of B7 molecules and their APC function. By using macrophages from congenic mice resistant or susceptible to infection with T. gondii, the influence of genetic background on the expression of CD86/B7-2 by parasitized macrophages was tested. A strain-dependent regulation of this co-stimulatory molecule was compared with the concomitant parasite-triggered cytokine responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Media, reagents and antibodies
IMDM (Gibco, Eggenstein, Germany) supplemented with 2 mM L-glutamine, 50 µM 2-mercaptoethanol and 5% heat-inactivated FCS (Gibco) was used in cell culture and proliferation assay, except for macrophage short-term culture which was performed using DMEM (Gibco) with 4.5 g/l glucose, 2 mM L-glutamine and 10% FCS. Purified protein derivative of tuberculin (PPD; Tuberculin `GT', 5000 IU/mg) was obtained from the Behringwerke (Marburg, Germany).

The following mAb were used: neutralizing anti-mouse IL-10 mAb JES5-2A5 from PharMingen (San Diego, CA), F4/80-specific mAb CI:A3-1 from Dianova (Hamburg, Germany), the phycoerythrin (PE)-labeled anti-CD80/B7-1 mAb 16-10A1, both PE-labeled and unconjugated anti-CD86/B7-2 mAb GL1 from PharMingen, and mouse IgG1 mAb GII which recognizes T. gondii surface antigen SAG1 from Biogenex (San Ramon, CA). Isotype-matched control mAb were from PharMingen and Dianova, FITC-conjugated F(ab')2 goat anti-rat and anti-mouse IgG respectively from Biozol (Eching, Germany).

Animals
Mice of the BALB/c and BALB.B strain had been obtained from the Jackson Laboratory (Bar Harbor, ME) and from Harlan Winkelmann (Borchen, Germany) respectively, and were bred under specific pathogen-free conditions. Their susceptibility to infection with T. gondii was controlled by i.p. injection with five cysts of mouse-avirulent strain DX. Consistent with previous observations (20), eight out of 10 BALB/c mice survived the chronic stage of infection whereas all BALB.B mice died.

Parasites and preparation of parasite antigen extract
Tachyzoites of mouse-virulent T. gondii strain BK were used and were propagated in L929 fibroblasts (ATCC, Rockville, MD) as described (19). Parasites were washed twice in PBS (600 g, 15 min). Soluble T. gondii antigen (sTgAg) was prepared from 109 purified tachyzoites by using a French press, and contained 10 mg protein and <0.5 pg/ml LPS. Cell lines and parasites were routinely tested for mycoplasma contamination.

Macrophage preparation and challenge by T. gondii
Peritoneal macrophages were prepared from >3-month-old male or female mice either 4 days after i.p. injection of sterile 3% thioglycolate or 2 days after i.p. infection with 103 toxoplasma and control injection of PBS respectively. When testing macrophages from BALB/c and BALB.B mice in parallel, the cells were prepared from sex- and age-matched animals. Up to 107 cells were plated with 10 ml medium containing additional 30% supernatant of L929 cells into untreated Petri dishes (Greiner, Nürtingen, Germany). After 24 h of incubation at 37°C, non-adherent cells were removed and adherent cells were harvested using a cell scraper. This protocol results in a population of >94% F4/80+ macrophages regardless of the pre-treatment. After washing, macrophages from thioglycolate-treated mice were used in T cell stimulation assays. Alternatively, macrophages from infected, PBS- or thioglycolate-treated mice were cultured with macrophage medium for a further 24 h before being subjected to flow cytometric analysis or RNA preparation. During that time, aliquots of thioglycolate-elicited macrophages were exposed to sTgAg or a 2-fold number of live toxoplasma, in some experiments in the presence of additional anti-IL-10 mAb JES5-2A5 or irrelevant rat IgG1 (2.5 ng/ml). As controlled by Trypan blue exclusion, >95% of cells from macrophage–parasite co-cultures were viable and in >80% of them intracellular toxoplasma were detectable 24 h post-inoculation by immunostaining performed as described (21).

Measurement of intracellular toxoplasma proliferation
In microtiter culture, thioglycolate-elicited macrophages (105/200 µl) were incubated with titrated numbers of toxoplasma. After 16 h, cultures were pulsed with 37 kBq [3H]uracil for a subsequent 24 h. The amount of [3H]uracil uptake directly corresponding to the parasite growth (22) was measured by liquid scintillation counting. Results are expressed as mean c.p.m. ± SD of triplicate test cultures.

Measurement of antigen-dependent T cell stimulation
For T cell proliferation assay, irradiated (20 Gy) macrophages (5x103/well) of BALB/c origin were co-cultured in flat-bottom A/2 microtiter plates (100 µl/well) with syngeneic LNC.2 (23) Th1 cells (104/well) and toxoplasma (1670/well) in the presence or absence of PPD (100 µg/ml). Parallel cultures were not inoculated. Anti-CD86/B7-2 mAb GL1 or isotype control mAb (100 µg/ml) was added. After 2 days of incubation, test cultures were pulsed with 7.4 kBq/well [3H]thymidine (Amersham-Buchler, Braunschweig, Germany) for a further 20 h and then processed as described for measurement of parasite proliferation.

Flow cytometry
Cells (5x105/sample) were washed and resuspended for 30 min at 4°C with the indicated mAb or, for double staining, with antibody mixtures. For detection of intracellular parasites, cells had been permeabilized by saponine using the Cytofix/Cytoperm kit (PharMingen). Samples containing unlabeled primary antibody were washed again and incubated with the FITC-conjugated secondary reagent. After final washes, cells were fixed with 0.7% paraformaldehyde. Samples were gated on macrophages based on forward and side scatter and controlled by F4/80 staining. Per sample, 104 macrophages were collected on a FACScan (Becton Dickinson, Heidelberg, Germany) and analyzed using Lysys II software. The flow cytometer was calibrated weekly and compensation was adjusted to minimize interference of PE and FITC fluorescence signals.

Detection of CD86 gene transcripts by RT-PCR
Total RNA was extracted by the guanidinium thiocyanate method from 4x106 macrophages or 107 T. gondii parasites. Reverse transcription of poly(A)+ mRNA was performed using oligo(dT) primers and MMLV reverse transcriptase from Clontech (Palo Alto, CA). Synthesized cDNA was used as template in subsequent PCR. Sequences of gene-specific primers and internal probes were: for the analysis of CD86 5'-GGGGGATCCATGGGCTTGGCAATCCTTAT-3' (sense), 5'-TCGGGTGACCTTGCTTAGACGTGCAGG-3' (antisense) and 5'-TGAACATTGT-GAAGTCGTAGAGTCCAGT-3' (probe), of T. gondii marker SAG1 5'-GCCGTTGTGCAG-CTTTCCGTTCTTC-3' (sense), 5'-ATCCCCCGTCCACCAGCTATCTTCT-3' (antisense) and 5'-GCGCGTCTCACTGCCTTCGGAAACATACTC-3' (probe), the housekeeping glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene expression was detected using primers from Clontech. PCR was carried out by incubation of the reaction mix in 28 cycles as follows: 1 min 95°C/1 min 60°C/1 min 72°C. The PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining and visualized with UV.

Homology of amplificates to the respective CD86 or SAG1 sequences was controlled by Southern blot analysis. Gels were denatured for 45 min in 0.5 M NaOH, 1.5 M NaCl, neutralized for 30 min in 0.5 M Tris–HCl, 3 M NaCl, pH 8 and blotted overnight to Nytran membrane (Schleicher & Schüll, Dassel, Germany) according to the standard protocol (24). The membrane was then UV cross-linked (0.6 J/cm2) and blots were prehybridized at 68°C in 5xSSC, 1% Blocking Reagent (Boehringer, Mannheim, Germany), 0.1% N-lauroyl-sarcosine and 0.02% SDS for 1 h. For hybridization, the corresponding oligonucleotide probes were 3'-end-labeled with digoxigenin using a DIG Oligonucleotide 3'-End Labeling kit (Boehringer). Hybridization was performed overnight at 54°C in the prehybridization buffer. Then, the membranes were washed twice in 2xSSC, 0.1% SDS for 5 min and twice in 0.1xSSC, 0.1% SDS for 5 min at 54°C. Subsequent immunodetection with alkaline phosphatase and the chemiluminescence substrate CSPD was carried out by using the DIG Luminscent Detection kit (Boehringer) according to the manufacturers instructions.

Cytokine secretion assays
Supernatants from cultures (3x105 cells/ml) parallel to those used for macrophages phenotyping or mRNA analysis were collected 24 h post-inoculation,and IL-1{alpha}, IL-10, IL-12(p40 + p70) and TNF-{alpha} were quantified using specific sandwich ELISA from Genzyme (Cambridge, MA). Recombinant mouse cytokines (Genzyme) served as reference standards. Assays had a minimum sensitivity of 5 (IL-1{alpha}, IL-12 and TNF-{alpha}) or 1 (IL-10) pg/ml.

Statistics
The unpaired Student's t-test and the Mann–Whitney U-test were performed, and P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
T. gondii triggers up-regulation of CD86 expression in macrophages infected in vitro or in vivo
Parasitized macrophages promote higher proliferation of T. gondii-specific mouse CD4+ T cell clones than macrophages exposed to dead toxoplasma or parasite extract as a source of antigen. Based on this observation, expression of B7 co-stimulatory molecules was analyzed on peritoneal macrophages following experimental infection. After a 24 h challenge of BALB/c inflammatory macrophages with live toxoplasma at a 2:1 parasite:host cell ratio, cell surface expression of CD86/B7-2 is selectively up-regulated in comparison to untreated cells (Fig. 1Go): the percentage of CD86+ cells slightly increases combined with a 29% increase in mean fluorescence intensity, whereas surface expression of CD80/B7-1 is not significantly altered (Fig. 1BGo). In comparison, resident peritoneal macrophages show no substantial shift in CD86 MFI values when challenged: 17.2 ± 3.5 versus 20.1 ± 4.0 (P = 0.32), indicating a need for macrophage preactivation as recently shown on T. gondii-triggered IL-12 production (16). CD86 up-regulation further depends on the presence of live parasites since administration of heat-killed toxoplasma proved to be ineffective (not shown).



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  		Fig. 1. Expression of B7 co-stimulatory molecules on macrophages challenged with T. gondii. Thioglycolate-elicited peritoneal macrophages from BALB/c mice were cultured in the presence or absence of a 2-fold number of live toxoplasma. After 24 h, cells (viability >=96%) were harvested and stained for cytofluorimetric analysis with either mAb CI:A3-1 (anti-F4/80) and FITC-conjugated anti-rat IgG, or PE-labeled mAb 16-10A1 (anti-CD80) or PE-labeled mAb GL1 (anti-CD86) or isotype-matched irrelevant mAb as control. (A) Results from one representative experiment showing the respective control staining (open histogram) and specific mAb binding (dark histogram) with the percentage of positive cells. (B) Mean fluorescence intensities (MFI) for CD80 and CD86 expression on cells from untreated (open bars) and T. gondii-treated (hatched bars) macrophages cultures. Values are means ± SD from six experiments, *P = 0.004.

 
T. gondii-induced up-regulation of CD86 on macrophages was confirmed at the mRNA level. As shown by RT-PCR, the amount of CD86 transcript is enhanced in peritoneal macrophages from T. gondii-inoculated versus untreated cultures (Fig. 2Go). This difference is most significant in macrophages isolated from PBS-injected mice. In comparison, thioglycolate-elicited cells produce a strong CD86 mRNA signal already without parasite challenge. The parasitized status of the cells was controlled by additional detection of transcripts for toxoplasmic marker SAG1. Similar analyses were performed using macrophages isolated from toxoplasma-infected mice and demonstrate that CD86 expression is strongly up-regulated also following in vivo challenge by the parasite (Fig. 2Go).



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  		Fig. 2. Induction of macrophage CD86 gene expression by T. gondii. Total RNA was extracted from BALB/c peritoneal exudate macrophages (lanes 1, 3, 4, 6 and 7) or T. gondii parasites (lane 2), subjected to reverse transcription and amplified by PCR using the indicated primers. Amplificates specific for CD86 or toxoplasmic SAG1 were detected by Southern blot. Macrophages had been isolated 2 days after i.p. PBS injection and were left untreated (lane 1) or were incubated with a 2-fold number of live parasites (lane 3) during subsequent 24 h culture. In parallel, macrophages were harvested 2 days after i.p. infection of mice with toxoplasma (lane 4). Lane 5 represents the H2O negative control, M the molecular size markers. For comparison, macrophages had been isolated from thioglycolate-treated mice and were incubated with toxoplasma (lane 7) or without (lane 6). As a control for equal amplification and gel loading, the constitutively expressed G3PDH gene message was analyzed simultaneously (upper panel).

 
Toxoplasma-induced up-regulation of CD86 in macrophages enhances their ability to promote antigen-directed T cell proliferation
In order to test whether the increase in cell surface expression of CD86 has functional consequences, parasitized macrophages were used as APC in T cell stimulation assays. As determined via the proliferative response of PPD-specific CD4+ T cells, T. gondii-infected macrophages are more efficient T cell stimulators than macrophages from uninfected control culture (Fig. 3Go). Up-regulated expression of CD86 contributes to the augmented APC function of parasitized macrophages since inhibition of CD86 ligand interaction by neutralizing antibody reduces their T cell stimulatory capability to a level similar to that of uninfected cells (Fig. 3Go). A complete reduction was not attained, suggesting that the test macrophages may provide additional co-stimulatory signals besides CD86. Consistent with this, blocking of CD86 in uninfected cells produced only a marginal effect on their T cell stimulatory capability.



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  		Fig. 3. CD86 dependency of antigenic T cell activation by T. gondii-infected macrophages. Cells of a PPD-specific CD4+ T cell line were incubated with syngeneic BALB/c peritoneal exudate macrophages and antigen in the presence or absence of live T. gondii parasites (open bars). Parallel test cultures additionally contained anti-CD86 mAb GL1 (black bars) or isotype control mAb (cross-hatched bars) at a concentration of 100 µg/ml. Proliferation was measured via [3H]thymidine incorporation during the last 18 h of a 3 day assay. Values represent mean c.p.m. ± SD from triplicate test cultures with PPD, controls without antigen yielded <120 c.p.m., those without T cells <151 c.p.m. Significant differences (P < 0.05) were observed between uninfected and toxoplasma-infected test cultures, and in the latter between anti-CD86 and control mAb-treated ones.

 
In BALB/c macrophages challenged by T. gondii, up-regulation of CD86 correlates with the intracellular parasite load whereas equally infected BALB.B macrophages exhibit a converse CD86 response
In the preceding experiments, the macrophages analyzed were from BALB/c mice which are resistant to infection with T. gondii. Incubation of macrophages from susceptible BALB.B mice with T. gondii parasites resulted in a down-regulation of CD86. To exclude the possibility that different rates of infection in toxoplasma-inoculated BALB/c and BALB.B macrophages may account for such discrepancy, the cells were double-stained for surface CD86 and intracellular parasites. After a 24 h challenge with live toxoplasma, staining of T. gondii marker SAG1 revealed comparable parasite loads with 82 versus 83% parasitized BALB/c and BALB.B macrophages respectively (Fig. 4Go). Accordingly, a similar intracellular parasite growth was measured in both macrophage populations when co-cultured with titrated numbers of toxoplasma (Fig. 5Go). Infection of BALB.B macrophages is accompanied by a decrease in CD86 surface expression, whereas parasitized BALB/c macrophages react with an up-regulation of CD86 (Fig. 4Go). Here, the highest density in CD86 expression was detected on cells which simultaneously bear maximum amounts of the parasite marker. These findings confirm, at the single-cell level, our previous results and indicate a correlation between the degree of infection and CD86 up-regulation in macrophages host cells from BALB/c mice.



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  		Fig. 4. CD86 expression on T. gondii-infected BALB/c and BALB.B macrophages in relation to the intracellular parasite load. Peritoneal exudate macrophages isolated from BALB/c and BALB.B mice were cultured for 24 h in the presence or absence of live T. gondii parasites. Then, cells were harvested and surface stained with mAb GL1–PE or PE–isotype control and intracellularly with mAb GII or an irrelevant mAb plus FITC-labeled anti-mouse IgG. In cytofluorimetric analysis, samples were gated according to the background staining with isotype control mAb and the percent distribution of cells in all quadrants is indicated. Results from one representative experiment of three are shown.

 


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  		Fig. 5. T. gondii proliferation in BALB/c and BALB.B macrophages. Peritoneal exudate macrophages from BALB/c (dark squares) and BALB.B (light squares) mice were incubated at varying ratios with live T. gondii parasites. Intracellular parasite growth was measured via uptake of [3H]uracil during the last 24 h of 40 h co-culture. Values represent mean c.p.m. ± SD from triplicate test cultures, controls lacking parasites yielded <1840 c.p.m..

 
In BALB/c versus BALB.B macrophages, T. gondii triggers a biased secretion of IL-1{alpha} and TNF-{alpha} or IL-12 and IL-10
As an alternative readout to control whether infected BALB.B macrophages react to the parasite, their secretory response was analyzed. A parasite-triggered secretion of transforming growth factor-ß, IL-12p40, TNF-{alpha} or IL-10 has been demonstrated on mouse peritoneal macrophages or microglial cells (17,19,25). Preliminary data indicated that a 24 h duration of macrophage co-culture with a 2-fold number of parasites allows reliable detection of IL-1{alpha}, IL-10, IL-12 and TNF-{alpha} in the supernatant. The concentrations peaked differently between 24 and 72 h post-inoculation but host cell lysis was apparent. As revealed by ELISA using 24 h supernatant, both BALB/c and BALB.B macrophages are stimulated by T. gondii to produce cytokines; however, disparate secretion profiles were observed (Fig. 6Go). While uninfected macrophages from both strains produce all cytokines at the lower limit of detection, T. gondii-infected BALB/c macrophages secrete substantial amounts of IL-1{alpha}, IL-12(p40 + p70) and some TNF-{alpha}, those of BALB.B origin preferentially IL-10 and significantly more IL-12. This pattern was reproduced in 48 h co-cultures containing equal numbers of macrophages and parasites.



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  		Fig. 6. Profiles of T. gondii-triggered cytokine secretion in BALB/c (dark bars) and BALB.B (light bars) macrophages. Peritoneal exudate macrophages from either mouse strain were cultured for 24 h with live toxoplasma at a parasite:host cell ratio of 2:1 or were left untreated. Supernatants were assayed for cytokine production by ELISAs specific for IL-1{alpha}, TNF-{alpha}, IL-12(p40 + p70) or IL-10. Values are the means ± SD from two independent supernatant productions, differences in cytokine production between parasitized BALB/c and BALB.B macrophages are significant (P < 0.05). Results from one representative experiment of three are shown.

 
In order to exclude a possibly different impairment in viability of BALB/c and BALB.B macrophages due to parasite replication, the cells were alternatively treated with sTgAg. Such antigen extract has been shown to stimulate release of IL-12p40 in thioglycolate-elicited peritoneal macrophages (26). Quantitation of IL-12(p40 + p70) in 24 h-supernatants revealed dose-dependent responses in both macrophages and confirmed a 4- to 5-fold higher IL-12 production by BALB.B macrophages (Fig. 7Go). In T. gondii lysate, two monokine-inducing activities have been distinguished which preferentially trigger IL-1ß and TNF-{alpha} or IL-10 and IL-12p40 gene expression (26). In that context, our data suggest that a different parasite activity predominantly triggers the host cell response in BALB/c and BALB.B macrophages respectively.



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  		Fig. 7. IL-12 secretion triggered by sTgAg in BALB/c (dark bars) and BALB.B (light bars) macrophages. As described in Fig. 6Go, cells were stimulated by 24 h incubation with titrated doses of sTgAg or remained untreated. Supernatants were assayed for IL-12(p40 + p70) by ELISA. Values are means ± SD from three supernatant productions. Results from one representative experiment of four are shown.

 
In BALB.B macrophages, parasite-triggered release of IL-10 causes down-regulation of CD86
A possible interference of T. gondii-induced IL-10 secretion and concomitant modulation of CD86 surface expression was analyzed in BALB.B macrophages. Addition of anti-IL-10 mAb antagonizes down-regulation of CD86 following toxoplasmic challenge (Fig. 8Go). The effect is specific since an irrelevant control mAb failed to influence CD86 expression on these macrophages. Thus, evidence is provided for a feedback inhibition of the CD86 response in T. gondii-infected macrophages via autocrine or paracrine IL-10.



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  		Fig. 8. IL-10-regulated cell surface expression of CD86 on BALB.B macrophages upon challenge by T. gondii. Peritoneal exudate macrophages were cultured with a 2-fold number of live toxoplasma in the presence of neutralizing anti-IL-10 mAb JES5-2A5 or isotype control mAb. Control cultures remained untreated. After 24 h, cells were harvested and stained for cytofluorimetric analysis of CD86 as described in Fig. 1Go. Results expressed show the respective control staining (open histogram) and specific mAb binding (dark histogram) with the percentage of positive cells (left no.) and their mean fluorescence intensity (in italics) from one representative of two experiments. Similar results were obtained on cells cultured with toxoplasma in the presence or absence of the control mAb.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
T. gondii-induced host cell activation is supposed to play a role in the initiation of immune resistance and may influence intracellular development of the parasite. One acute reaction of mouse macrophages and brain glial cells is the expression and secretion of cytokines triggered by live toxoplasma or parasite lysate (16,17,19,25). Although the causative molecules and the mechanisms of cellular responsiveness are still unclear, recent data indicate that both T. gondii bradyzoites and tachyzoites provide such stimuli (19) and that macrophages are activated by at least two biochemically distinct parasite factors (26).

The results of the present study establish CD86/B7-2 as a further immunofunctional parameter which underlies toxoplasmic modification of macrophages host cells. Surface expression of CD86, but not of CD80, proved to be enhanced in BALB/c macrophages upon toxoplasma infection, which leads to an increased co-stimulatory activity in presenting foreign antigen. By selectively triggering CD86, the effect of T. gondii resembles that produced by Trypanosoma cruzi in macrophages (27). In comparison, the effect of challenge with Leishmania donovani is a decreased expression of CD80 and heat-stable antigen (28,29). As recently shown, T. gondii triggers a different reaction in human monocytes by up-regulating surface expression of CD86 and inducing that of CD80 (30). On these cells, CD86 is up-regulated by the parasite via an indirect, IFN-{gamma}-dependent pathway (31). In T. gondii-infected mouse macrophages, CD86 up-regulation was significant only in thioglycolate-elicited cells suggesting a need for macrophage preactivation as already found for their IL-12 response (16). Up-regulation of macrophage CD86 is accompanied by an increase in CD86 gene message indicating that parasite stimulation affects the transcriptional level. Expression of CD86 can be induced by external cytokine signals; however, strong evidence for a direct effect mediated by the parasite on its host cell is obtained from the simultaneous detection of intracellular toxoplasma and enhanced surface CD86 in single macrophages. In in vivo studies, CD86/B7-2 has been shown to be the major CD28 co-stimulatory ligand active in clonal expansion of antigen-specific T cells (reviewed in 32). Our findings that T. gondii-triggered up-regulation of CD86 in macrophages (i) indeed augments their T cell stimulatory activity, and (ii) occurs following experimental infection in vivo suggest a relevance in the establishment and maintenance of a parasite-directed T cell response.

Macrophage CD86 response to toxoplasmic challenge varies with the MHC genotype with a significant up-regulation being observed in BALB/c macrophages, whereas surface expression of CD86 decreases in BALB.B macrophages. In addition, contrasting profiles of parasite-triggered cytokine secretion were detected: IL-1{alpha} plus moderate IL-12 in BALB/c versus il-10 plus high IL-12 in BALB.B macrophages. Evidence from anti-IL-10 treatment of T. gondii-infected BALB.B macrophages indicates an interference of their cytokine response with CD86 expression resulting in a down-regulation of CD86. Thereby, macrophage functions essential in innate and adaptive immunity are regulated by the parasite dependent on the MHC of the host. Since the two congenic mouse strains are resistant or susceptible to infection with T. gondii, our findings in vitro imply a significance to the in vivo situation. In inbred and mutant mice, the presence of the d allele at the MHC class I L locus strictly correlates with resistance (15), the H-2b haplotype predisposes to susceptibility (33). Thus, genetic differences in the acute reaction of parasitized host cells, as demonstrated here, are associated with the outcome of infection.

In view of protective effects that have been attributed in mouse models of T. gondii infection to IL-1 or IL-10 or IL-12 (34–36), the cytokine mixture released from parasitized cells may be pivotal in shaping the immune response to the pathogen. According to this hypothesis, a genotype-determined massive production of IL-12, as proves characteristic of the host cell reaction in BALB.B macrophages, may cause the aberrant and lethal Th1 response in the intestine of perorally infected mice with the H-2b haplotype (37). BALB/c mice do not develop this pathology (37) but protective activity of IL-12 during early infection has been detected by in vivo neutralization (38). Interestingly, the profiles of cytokine secretion by parasitized BALB/c and BALB.B macrophages are congruent with the preferential induction of IL-1ß plus TNF-{alpha} and IL-12 plus IL-10 respectively by protease-resistant or sensitive constituents in parasite lysate (26). In combination, both findings suggest MHC-determined sensitivities for either activation pathway. From this model of dichotomous activation of macrophages by T. gondii (Fig. 9Go), a hyper-reactivity to the IL-12/IL-10-inducing parasite factor(s) can be expected for cells of the H-2b genotype.



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  		Fig. 9. The double dichotomy in macrophages acute reaction to T. gondii. Two activities of tachyzoite lysate as distinguished biochemically by Grunvald et al. stimulate murine inflammatory macrophages to preferentially express mRNA transcripts for IL-1ß and TNF-{alpha} or IL-12p40 and IL-10 (26). That dichotomy is mirrored in the MHC-dependent profiles of parasite-triggered cytokine secretion and regulation of CD86 as revealed by the present study.

 
In conclusion, the present data extend the concept of functional host cell modification by T. gondii by the facets of its MHC dependency and impact on macrophage CD86/B7-2 expression. First evidence is provided for a role of the MHC in the cytokine response of parasitized host cells. The striking correlation between MHC-dependent differences in parasite regulation of host cell functions and susceptibility or resistance of donor mice to T. gondii argues for a key role that parasitized macrophages will play in promoting immunopathology in H-2b or protection in H-2d mice.


   Acknowledgments
 
We are grateful to U. Bonifas and K. Buchholz for excellent help at FACS and PCR analyses, E. Schmitt and H. M. Seitz for providing cells and T. gondii strains, and G. Reichmann and C. MacKenzie for critical comments. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 194/B11). R. D. is recipient of a DFG doctoral fellowship.


   Abbreviations
 
APCantigen-presenting cell
G3PDHglyceraldehyde-3-phosphate dehydrogenase
PEphycoerythrin
PPDpurified protein derivative
sTgAgsoluble T. gondii antigen
TNFtumor necrosis factor

   Notes
 
Transmitting editor: S. H. E. Kaufmann

Received 5 March 1998, accepted 9 November 1998.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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