Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association between susceptibility and type 2 cytokine production

Cytokine production in the spleens of mice infected with the protozoan parasite Trypanosoma cruzi was analyzed in three models which differ in the outcome of the infection. Using immunocytochemical techniques to detect cytokine-producing cells, the production of type 1 [interleukin-2 (IL-2) and interferon (IFN)-γ], type 2 (IL-4, IL-5, IL-10), inflammatory [tumor necrosis factor (TNF)-α, IL-1α, IL-6] and regulatory (transforming growth factor-β) cytokines were examined. With the exception of IL-4 and IL-5, cells producing all of the cytokines assayed were detected in both the resistant and susceptible models of T. cruzi infection. Cells producing IL-4 and IL-5 were not detected until later in infection in the resistant mice (>34 days), at about the time animals of the susceptible strain succumb to the infection. Mice of the susceptible model showed a slight delay in the appearance of cells producing the type 1 cytokines IL-2 and IFN-γ and an earlier appearance of TNF-producing cells, in comparison to resistant mice. Cells producing IL-2 or IL-10 were transient in their appearance in the spleen while cells producing IL-1, IL-4, IL-5, IL-6, IFN-γ, TNF, or TGF-β were first detectable in either the acute or post-acute stage of the infection and persisted up to 700 days post infection in two different resistant models of the infection. Cells producing IFN-γ, TNF-α and TGF-β were particularly numerous even very late in the infection. Double-staining techniques were used to show that the vast majority of the IFN-γ-producing cells in the spleen were CD4^?, CD8^? α/β TCR⁺ T cells. This study confirms the transience of IL-2 production in the acute stage of T. cruzi infection and the persistent and simultaneous production of type 1 and type 2 cytokines during the late-acute and chronic stages of the infection. Susceptibility or resistance to T. cruzi infection does not associate with a Th2 pattern of cytokine production in the three models examined in this study. The overlapping pattern of type 1 and type 2 cytokine-producing cells in both the acute and chronic stages of T. cruzi infection demonstrates that longterm infections do not necessarily lead to a dominance of either type 1 or type 2 cytokine production.     

Localization of NPY-immunoreactivity in the cat's visual cortex

Summary Using a polyclonal antiserum against neuropeptide Y (NPY; J.M. Allen et al. 1983a) immunohistochemistry was carried out using the PAP method. Neurones displaying NPY-like immunoreactivity are seen mainly in cortical layers V/ VI, adjacent white matter and corona radiata. Only few neurones occur in superficial layers II/III. Neurones are multipolar to bitufted with spineless dendrites; somata are either round (layers V, II/III) or spindle-like (layer VI, white matter) with diameters between 16 and 20 m. Axones were identified by their initial smoother profiles, which are smaller in diameter than principal dendrites, by their typical branching pattern and the occurrence of terminal portions. It was found, that the degree of axonal ramification in proximal parts of axones is rather poor. Most NPY-neurones seem to project intracortically or even locally, except neurones in layers VI and the white matter. The latter neurones have ascending axonal branches terminating in layer VI and V, thus contributing to the dense NPY-plexus in these layers, whereas some layer VI neurones have axonal branches descending into the white matter. The axonal plexus in upper cortical layers is most probably fed by the ascending axones of layer V neurones, passing layer IVc in a strictly vertical direction. Fine smooth fibers of unknown origin which ascend from the white matter in a vertical direction through the grey matter also contribute to the plexus in layer II/III. In semithin sectioned material three terminal types were identified. Firstly, en passant boutons on immunnegative pyramidal neurones, secondly, perisomatically arranged, basket-like terminals, bending around unstained non-pyramidal neurones, and thirdly, about 60 m long vertically oriented rows of boutons exclusively on apical dendrites of layer II/III pyramidal neurones. Due to the unconspicious axonal pattern and the frequently observed basket-like terminal form, we conclude that most NPY-ir neurones can be regarded as a class of unspecific local field basket cells; the origin of the vertically arranged bouton rows has been yet to determined.     

Differential cytokine profile in children with cystic fibrosis

The previously observed occurrence of antineutrophil cytoplasmic autoantibodies (ANCA) in patients who have cystic fibrosis (CF), together with the reported decrease in IgG2, a Th1-controlled isotype, suggests a potential for Th1/Th2 imbalance in CF patients with a possible Th2 predominance. 48 CF patients and 16 controls had levels of IFNgamma, IL-4, and IL-10 measured in supernatants of whole blood cell cultures stimulated by lipopolysaccharide (LPS) and phytohemaglutinine (PHA). The patients were divided into 2 groups: "low responders", having negligible secretion of cytokines (IFNgamma: 10.0-200.0 pg/ml, IL-4: 0.0-0.3 pg/ml) and "high responders", producing high levels of both IFNgamma (500.0-2000.0 pg/ml) and IL-4 (1.0-200.0 pg/ml). There was a statistically significant (P < 0.01) deterioration of lung function measured by an FEV(1) decline by 11.2% over 3 years in the "low responder" group. 10 of 16 "low responders" had chronic lung infections with P. aeruginosa while such infection was less prevalent in the "high responder" group where only 13 of 32 CF patients had positive cultures. A shift towards Th2 response was observed in the "high responder" group as children chronically infected with P. aeruginosa had greater IL-4 production than non-infected CF patients within the same cohort. ANCA autoantibodies were found only in the "high responder" group. Th2 immune response predominance in a subset of CF patients is associated with chronic P. aeruginosa infection.     

The role of IL-17 in systemic lupus erythematosus and its potential as a therapeutic target

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies production and immune complex deposition with systemic clinical manifestations. Interleukin (IL)-17-producing cells play a crucial role in disease pathogenesis and represent an attractive therapeutic target.

Areas covered: This review provides an update on the possibility of targeting IL-17 in SLE. The rational for this approach as well as currently available and future targets are discussed.

Expert opinion: Although human expression studies and animal models indicate that IL-17 blocking may be a promising therapeutic strategy for SLE, direct evidence for IL-17 inhibition in SLE patients is unavailable. Biologic therapies and small-molecule drugs that target IL-17 production are required for the achievement of a favorable clinical effect in SLE patients.     

Reciprocal heterotopic callosal connections between the two striate areas in Tupaia

Summary WGA-HRP injections were placed into area 17 close to the border with area 18 of Tupaia belangeri in order to study the callosal connections of the striate area in this animal. Most callosal neurons were found in the striate cortex (57.6–86.9%), some in the extrastriate area 18 (10.6–28.1%), and a few in even more temporal regions (2.5–14.3%). Concerning only the area 17, reciprocal homotopic connections could be observed as a strip along the area 17/18 border. Additionally, heterotopic callosal connections could be seen in regions representing the binocular visual field, especially the lower part. The area 17 cells were mostly located in the supragranular layers II and III (94.1–97.2%). But neurons could also be found in the infragranular layers, especially layer VI (2.6–5.2%) and in layer IV (0.2–1.1%). Homotopic projections were mostly seen in layers IIIc and V. The majority of the supragranular and infragranular neurons are pyramidal cells. However, a newly defined subpopulation of neurons, most probably stellate cells, were discovered forming a band in sublayer IIIc, very close to the layer III/IV border.     

Complex pattern of Th1 and Th2 activation with a preferential increase of autoreactive Th1 cells in BALB/c mice with proteoglycan (aggrecan)-induced arthritis

The central role of CD4+ T cells and the balance between T helper (Th) subpopulations in the pathogenesis of autoimmune diseases have been extensively studied. Proteoglycan (aggrecan)-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA), which is characterized by a Th1 dominance at the onset of the disease. In addition to CD4+ T cells, antigen-presenting B cells and autoantibodies seem to play an important role in the development and regulation of PGIA. To identify proteoglycan-specific CD4+ T cell subsets and Th1- and Th2-supported antibody isotypes during the progression of PGIA, spleen cells of proteoglycan-immunized BALB/c mice were harvested at different times of immunization, and at different stages of the disease, and their cytokine production and antigen-specific antibody isotype profiles were determined by enzyme-linked immunospot (ELISPOT) assays. Both Th1 and Th2 cytokine-producing cells, with the predominance of IL-4/IL-5-secreting cells, were detected during the prearthritic stage, and a shift toward a Th1 dominance was observed at the time of onset of arthritis. Tissue homogenates of acutely inflamed joints contained significantly higher levels of interferon-gamma than IL-4. The prearthritic period and both the acute and chronic phases of joint inflammation were characterized by IgG1 dominance in the sera and this correlated with the number of IgG1-secreting B cells in the spleen. However, the ratio of autoreactive IgG1/IgG2a-secreting cells decreased in arthritic animals. These results indicate the activation and possible regulatory roles of both Th1 and Th2 subsets in the autoimmune process, with the necessity of a relative increase of autoreactive Th1 cells for the induction of joint inflammation.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

Direct and indirect role of Toll-like receptors in T cell mediated immunity

Toll-like receptors (TLR) are pathogen-associated molecular patterns (PAMPs) recognition receptors that playan important role in protective immunity against infection and inflammation.They act as central integrators ofa wide variety of signals,responding to diverse agonists of microbial products.Stimulation of Toll-like receptorsby microbial products leads to signaling pathways that activate not only innate,but also adaptive immunity byAPC dependent or independent mechanisms.Recent evidence revealed that TLR signals played a determiningrole in the skewing of na(?)ve T cells towards either Th1 or Th2 responses.Activation of Toll-like receptors alsodirectly or indirectly influences regulatory T cell functions.Therefore,TLRs are required in both immuneactivation and immune regulation.Study of TLRs has significantly enhanced our understanding of innate andadaptive immune responses and provides novel therapeutic approaches against infectious and inflammatorydiseases.Cellular & Molecular Immunology.2004;1(4):239-246.