Cytokine responses induced by Toxoplasma gondii in astrocytes and microglial cells.

To investigate the role of astroglia in intracerebral immune response to Toxoplasma gondii, astrocytes cultured from mouse brain were inoculated with mouse-virulent or -avirulent toxoplasma strains. In comparison to microglia/ brain macrophages, astrocytes as host cells allowed stronger proliferation of avirulent parasites. Toxoplasma infection of astroglia was accompanied by release of interleukin- (IL)1 alpha, IL-6, and granulocyte/macrophage colony-stimulating factor (GM-CSF) activity, whereas alternative challenge by lipopolysaccharide (LPS) evoked no IL-1 response and significantly higher titers of IL-6 and GM-CSF. At the mRNA level, both stimuli induced transcription of all three cytokines in astrocytes. Secretion of IL-1 and IL-6 upon infection was triggered by T. gondii brady- and tachyzoites in a time- and dose-dependent manner. Heat killing of parasites, but not an exposure to polymyxin B, abrogated their cytokine-inducing activity, thus indicating that an LPS-independent stimulus is provided by T. gondii. When administered in combination, LPS synergistically augmented the IL-1-inducing effect of toxoplasma infection. In comparison, T. gondii-induced, but not an LPS-triggered, IL-6 response of astrocytes resisted to antagonization with IL-10. The IL-6 response of parasitized astroglia was up-regulated by external tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta 1, with only TNF-alpha enhancing simultaneous release of IL-1. Substantial secretion of IL-10 and TNF-alpha was detected in T. gondii-infected microglia, but not in astrocyte cultures. A possibly autocrine stimulation of infected astroglia via IL-1 was found to be unlikely, since addition of IL-1 receptor antagonist did not affect the release of IL-6 and GM-CSF while inhibiting these responses in IL-1-treated cells. The findings substantiate a separate, T. gondii-induced pathway of astroglia activation characterized by the release of IL-1 which may drive local inflammatory reaction both at initial infection of the brain and during reactivating toxoplasmosis.

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain.

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in] parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.

Functional dichotomy of mouse microglia developed in vitro: differential effects of macrophage and granulocyte/macrophage colony-stimulating factor on cytokine secretion and antitoxoplasmic activity.

After differentiation either with exogenous macrophage (M) or with granulocyte/macrophage (GM) colony-stimulating factor (CSF) microglial cells were isolated from neonatal mouse brain cell cultures and were comparatively tested for secretory immune effector cell functions. Both factors obviously do not promote the development of cells with biased growth requirement; however, the two microglia populations displayed distinct potentials to produce inflammatory cytokines. Upon gradual stimulation by lipopolysaccharide, the cells harvested from M-CSF-driven culture released more interleukin-1 and tumor necrosis factor activity, GM-CSF-grown cells on the contrary proved superior in interleukin-6 secretion. This pattern was paralleled by corresponding different kinetics of cytokine release in both types of microglial cells. When infected with Toxoplasma gondii only GM-CSF-differentiated cells were able to restrict the intracellular multiplication of tachyzoites in the absence of external stimuli. As described for interferon-gamma-treated macrophages, the antiparasitic activity of this microglia population is due to the synthesis of reactive nitrogen intermediates, since it was antagonized by NG-monomethyl-L-arginine, a competitive inhibitor of the arginine-dependent metabolic pathway. Complementary to previous data which attest an intrinsic capability for antigen presentation to GM-CSF-grown microglia, the functional state of the cells elicited by M-CSF and GM-CSF, respectively, may correspond to the resting and an activated form of microglia as distinguished in vivo.

Differentiation driven by granulocyte-macrophage colony-stimulating factor endows microglia with interferon-gamma-independent antigen presentation function.

The antigen presentation function of microglial cells was analyzed after differentiation in neonatal mouse brain cell cultures supplemented either with macrophage (M) or granulocyte/macrophage (GM) colony-stimulating factor (CSF). The cells separated from concomitant astrocytes in both culture systems turned out to exhibit cytological characteristics of macrophages and bore MAC-1 and F4/80 markers in a similar way. When comparatively tested for accessory cell function, only microglia developed with GM-CSF were able to efficiently induce antigen-directed proliferation of a series of helper T cell lines representing both the TH1 and TH2 subtype. Antigenic T cell activation by this microglia population was performed without prior stimulation and exceeded that of M-CSF-dependently grown microglial cells, even if those had been pretreated with interferon-gamma (IFN-gamma). In contrast to such difference in function, low cell surface expression of MHC class II or intercellular adhesion molecule-1 determinants proved to coincide in both populations. Correlating with the capacity for antigen presentation, expression of membrane-bound interleukin-1 (IL1)--a costimulatory signal for TH2 cells--was augmented significantly in GM-CSF-grown microglia. In parallel, the interaction only of this microglia population with a selected TH1 cell line was accompanied by maximal release of T cell-stimulating factor, a cytokine recently identified as an IL1-analogous second signal for TH1 cells. Thus, a developmental process is suggested which produces a form of microglia specialized in antigen presentation and thereby acting uncoupled from IFN-gamma.

Extracellular matrix (ECM) modulates the EGF-induced migration of liver epithelial cells in serum-free, hormone-supplemented medium.

The influence of the extracellular matrix (ECM) glycoproteins collagen IV, laminin (LN), and fibronectin (FN) on the in vitro migration of epithelial cells was studied using the ECM migration track method (4) with preparations immunostained for LN and FN. The locomotion of rat liver epithelial cells stimulated to migrate in serum-free medium by epidermal growth factor (EGF) in the presence of insulin is inhibited by substratum-bound FN. The inhibition is concentration-dependent up to 0.7 microgram of the protein per cm2. Neither LN nor collagen IV decreased the number of migrating cells, indicating that the inhibition is a specific effect of fibronectin. The data also indicate that the FN-mediated inhibition of migration is an additional and not alternative mechanism to the well-established contact inhibition of locomotion (1) which also occurs in liver epithelial cell cultures. The system is being used for a further analysis of the factors that influence migration of normal and neoplastic epithelial cells and the biochemical mechanisms underlying the migration reaction.