Aleksandar Dujić

Interferon gamma alters the phenotype of rat thymic epithelial cells in culture and increases interleukin-6 production.

Rat thymic epithelial cells (TEC) in long-term culture were characterized by anticytokeratin monoclonal antibodies (mAbs) and electron microscopy. Phenotypic analysis performed by a large panel of mAbs showed that the highest percentage of these cells was of the subcapsular/medullary type. Recombinant rat interferon (IFN)-gamma up-regulated class-I and class-II MHC expression by TEC in culture as confirmed by immunohistochemistry and flow cytometry, but did not significantly alter other cell markers. TEC supernatants of IFN-gamma-treated cultures showed higher interleukin-6 (IL-6) activity, compared to the control, as determined by proliferation of the IL-6-sensitive B9-cell line. Increased IL-6 activity was probably not a consequence of increased TEC number in IFN-gamma-treated cultures because IFN did not significantly stimulate TEC proliferation in vitro. In contrast, IL-6 significantly stimulated TEC proliferation, indicating that this cytokine is not only a regulatory molecule for T-cell proliferation, but could also be an autocrine growth factor for thymic epithelium.

Interspecies differences in expression of cytokeratin polypeptides within thymic epithelium: A comparative immunohistochemical study

Cytokeratin (CK) polypeptide expression within the thymic epithelium of several mammalian species (mouse, rat, calf, pig, rabbit, and human) has been analyzed by the streptavidin-biotin immunoperoxidase method. Comparative analysis by a large panel of 17 monoclonal antibodies (mAbs) specific for individual CK polypeptides, pairs, or groups showed considerable heterogeneity of thymic epithelial cells (TEC) in each species. In addition, extreme interspecies difference in CK contents was observed. Four main phenotypic zones: the subcapsule/perivascular area, cortex, medulla, and Hassalls corpuscles (HC) were clearly identified, each characterized by different CK expression. Medullary TEC were more heterogenous and shared common CK polypeptides either with subcapsular/perivascular TEC, cortical TEC, or HC, in most species.

Ontogeny of Rat Thymic Epithelium Defined by Monoclonal Anticytokeratin Antibodies

Ontogenetic study on the expression of cytokeratin (CK) polypeptides within particular subsets of rat thymic epithelial cells (TEC) has been performed by a large panel of anti-CK monoclonal antibodies (mAbs) using the streptavidin-biotin immunoperoxidase method. Simultaneous presence of two or more CK subunits in the same TEC has been demonstrated by double immunoflouorescence labeling. The obtained results showed that the expression of CK polypeptides in fetal and neonatal thymus differed from the adult patterns. The main difference was observed in expression of CK10, 18, and 19 polypeptides. During fetal ontogeny, CK10 and 18 are markers for most medullary TEC or a subset of medullary TEC, respectively, whereas CK19 is mainly a pan-TEC marker. In the adult animals, they are localized in the cortical and a subset of medullary TEC (CK18), subcapsular/perivascular and some medullary TEC (CK19), or in a subset of medullary TEC and Hasall’s corpuscles (HC) (CK10). The switch in their expression in the cortex was observed during the first two weeks of postnatal life.

Thymic response to thermal injury in mice: I. Alterations of thymocyte subsets studied by flow cytometry and immunohistochemistry

The dynamics of thymocyte subset changes in mice subjected to sublethal thermal injury were studied in cell suspensions by flow cytometry and in situ by immunohistochemistry. Thermal injury caused acute thymic involution in the first 2 days which was the consequence of a considerable decrease in numbers of Thyl.2high+ CD4+ CD8+, cortical thymocytes. Medullary, Thyl.2low+ thymocytes were more resistant and their relative values increased. In the regenerative phase (2-14 days) the recovery of large CD4- CD8-, early thymocytes, mainly localized in the subcapsular area of the thymus, preceded the regeneration thymocytes of the cortical phenotype. Judged by the absolute numbers of medullary thymocytes it can be seen that CD4+ CD8- (T-helper/inducer cells) were more sensitive to the effect of thermal injury than CD4- CD8+ (T-suppressor/cytotoxic cells). While values of CD4+ CD8- cells were constantly and progressively lower during 2 weeks after thermal injury, absolute numbers of CD4- CD8+ cells showed cyclic changes with lower and higher values compared to controls. An increase in the numbers of CD4- CD8+ cells was found at day 6 after thermal injury.

Post-traumatic activation of draining lymph node cells. II. Proliferative and phenotypic characteristics.

Proliferative and phenotypic characteristics of cells in regional lymph nodes that drain burn injury were examined in rats on day 3 postburn, i.e. at the time of maximal spontaneous proliferation and of interleukin-2 and accessory cytokine (IL-1 and IL-6) production. The importance of IL-1 in spontaneous proliferation of draining lymph node cells was demonstrated by stimulation of IL-2-driven proliferation by recombinant IL-1 in vitro and by susceptibility of unstimulated proliferation to anti-IL-1 antibodies, while requirements for IL-6 in draining lymph node cell proliferation were less pronounced. Cell surface phenotyping revealed a slightly increased percentage of CD25+ cells in the blast cell population of freshly isolated draining lymph node cells after injury, which increased further during cultivation. Enrichment in CD8+ cells on day 3 following burn injury was demonstrated, while no changes in total cell population and CD4+ cells was noted. This was however preceded by pronounced percentual decrease of total T cells and CD4+ cells and by increases of B cells and MHC class II+ cells on day 1 postburn. Inhibition of draining lymph node cell proliferation by anti-MHC class II antibodies suggested that this proliferation was class II MHC dependent. The contribution of cell proliferation and/or cell influx to day 3 postburn draining lymph node cell activity is discussed.

Bidirectional Interactions between Thymocytes and Thymic Epithelial Cell Lines In Vitro

In vitro interactions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+CD8+ αβTCR10 thymocytes, induced apoptosis of a subset of these cells, and stimulhted proliferation of the BWRT (CD4-CD8- αβTCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+CD810 αβTCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC lines in vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.

Adhesion Molecules in the Thymic Microenvironment: Interactions between Thymocytes and Cloned Thymic Epithelial Cell Lines

Publisher Summary This chapter presents a study on the adhesion molecules, which participate in the interactions between thymocytes and cloned thymic epithelial cell (TEC) lines. Various subsets of TEC provide distinct stimuli to developing thymocytes in the thymus via direct cell–cell interactions and soluble molecules, in which various receptor–ligand pairs participate. In this study, two TEC lines (R-TNC.1 and TE-R 2.5) from long-term culture of the rat thymic epithelium were cloned. Based on detailed multimarker phenotypic analysis and electron microscopy, it was concluded that the R-TNC.1 line is a type of cortical TEC with nursing activity, whereas the TE-R 2.5 line belongs to the medullary TEC. By using an in vitro assay, it is shown that R-TNC.1 and TE-R 2.5 cell lines differently bind thymocytes and T cell hybridomas. Binding of thymocytes to both lines is mediated by LFA-1/ICAM-1, CD2, and Thy 1, but the extent of binding inhibition in the presence of specific monoclonal antibodies (mAbs) depends on TEC lines used. CD4 and CD8 as well as two novel molecules expressed on TEC, defined by 4D1 and 1D6 mAbs, are involved in adhesion of thymocytes to the medullary line.

17 – An Excess of IL-6 Production in the Early Muscle Stage of Trichinella spiralis Infection in Mice is Associated with Strain Susceptibility to Infection

Publisher Summary This chapter presents a study that investigates the difference in IL-6 production between BALB/c (H-2d) and C57B1/6 (H-2b) strains of mouse infected with Trichinella spiralis (TS). The study was performed three weeks after infection, when the phenomenon of autoantibody synthesis amplification was observed and when the final stage of parasite development commenced, accompanied by muscle inflammation. Female BALB/c and C57B1/6 mice of 8–10 weeks of age and 19–22 g body weight were used in the experiments. Mice of each strain were randomly divided into two groups containing 10 animals per group. They were exposed to TS infection (group TS) and a control group (C) of nontreated mice. Mice were sacrificed on day 21 for the determination of muscle larva recovery, production of specific antibodies, and analysis of IL-6 cytokine production. Infectious L1 larvae were obtained by digestion of minced TS-infected rat carcasses in 1% pepsin-HCl for four hours at 37° C. The mice were infected by oesophageal intubation with 200 L1 larvae each. The results show that BALB/c mice harbored approximately 50% fewer L1 larvae in their muscles than C57B1/6 mice. This confirmed previous findings that BALB/c mice are considered to be resistant and C57B1/6 susceptible with respect to muscle larva burden. A significant increase of IL-6 production by the antigen-stimulated spleen cells was found in the T. spiralis susceptible C57B1/6 mice. The increase of IL-6 production in resistant BALB/c mice did not reach statistical significance when compared to the controls, uninfected mice.

IL-1, TNF and IL-6 Release by Wound- inflammatory Cells During the Healing Process in Two Strains of Rats

Publisher Summary This chapter presents a study in which the cellular responses during the course of wound healing in two different inbred rat strains, Albino Oxford (AO) and Dark August (DA), are investigated. The rate of wound closure of full-thickness wounds, the type of wound-infiltrating cells, and the time course of interleukin 1(IL-1), interleukin 6 (IL-6), and tumor necrosis factor (TNF) production by wound-derived cells were measured by using a sponge–matrix model. Two round full-thickness wounds were prepared dorsally in both strains with a sharp, round metal blade of 1 cm diameter, and wound diameters were measured during seven days of the healing process. The study demonstrates that DA and AO rats differ in their healing capabilities. The DA rats showed an accelerated wound-healing course that was accompanied by a different pattern of wound cellular infiltration and significantly higher levels of IL-6 and TNF in supernatants of wound-derived cells compared to AO strain. A higher proportion of infiltrating lymphocytes and higher IL-6 and TNF activities in wound-cell supernatants could be one of the mechanisms that underline the accelerated wound-healing course seen in DA rats.

27 – Altered Functions of Peripheral Blood Mononuclear Cells and Granulocytes in Patients with Active Psoriasis

Publisher Summary This chapter presents a study on the altered functions of peripheral blood (PB) mononuclear cells and granulocytes in patients with active psoriasis, such as the mitogen and phorbol ester-induced proliferative lymphocyte response, as well as neutrophil activation, adhesion, and tumor necrosis factor (TNF) production. The cells were isolated from venous blood of 16 patients (13 men and three women) with severe and active generalized forms of psoriasis, and six healthy volunteers were included as controls. Peripheral blood cells were isolated by centrifugation on a Lymhoprep density gradient, and the mononuclear cells were washed and resuspended in RPMI supplemented with 5% fetal calf serum. Statistical analyses were made by using the Mann–Whitney test. The study found that the level of spontaneous lymphocyte proliferation did not differ between psoriatic patients and healthy controls. Phytohaemaglutinin (PHA)-induced lymphocyte proliferation showed a significantly lower level in patients with psoriasis compared to healthy controls. Spontaneous neutrophil TNF production did not differ between psoriatic patients and healthy controls, except that four out of 16 patients with psoriasis had extremely high levels. The results of the study demonstrated impaired peripheral blood lymphocyte proliferative response and altered functions of PB neutrophils in patients with active psoriasis.