Differential Effects of Male and Female Gonadal Hormones on the Intrathymic T cell Maturation

The study was undertaken to further elucidate a role of gonadal hormones in maintenance of normal thymocyte maturation and sexual dimorphism in the intrathymic T-cell development. Rats of both sexes were gonadectomized or sham-gonadectomized (controls) at age of 2 and 6 months, and 30 days later the thymus size, cellularity and thymocyte composition were evaluated. In both control and gonadectomized rats, in spite of age, sexual dimorphism in the thymus size and cellularity was found. Gonadectomy in 2-month-old rats of both sexes increased the thymus cellularity, volumes of both cortex and medulla and thymus size (to a less extent in males), while in 6-month-old rats, in this respect, it was effective only in females. In ovariectomized (OVX) rats the increase in volume of cortex was more marked in younger rats, while that of medulla did not differ between rats of different age. It seems obvious that in both groups of OVX rats the volume of medullary non-lymphoid component was enlarged (the increase in medullary volume was more pronounced than that in its cellularity). Unlikely, in rats orchidectomized (ORX) at age of 2 months the volume of this component was either decreased or unaltered (the increase in the volume of medulla was less conspicuous than that in the number of medullary thymocytes). In control and gonadectomized rats of both ages, sexual dimorphism in the composition of thymocyte subsets was also observed. Gonadectomy in 2-month-old rats affected distinct stages of thymocyte maturation in male (increased the relative proportions of CD4+8+TCRαβlow cells and their CD4–8+TCRαβlow precursors and decreased those of the most mature CD4+8-TCRαβhigh and CD4–8+TCRαβhigh cells) and female rats (decreased only the percentage of the least mature CD4–8-TCRαβ-cells). In older rats only ovariectomy had impact on the relative proportion of thymocytes decreasing, besides the relative proportion of CD4–8-TCRαβ- cells, those of CD4–8+TCRαβ-, CD4–8+TCRαβlow, positively selected CD4+8+TCRαβhigh and the most mature CD4+8-TCRαβhigh, CD4–8+TCRαβhigh cells and exerting an opposite effect on the percentages of CD4+8+TCRαβ- and CD4+8+TCRαβlow cells. Thus, results showed sex- and age-dependent changes in sensitivity of both the developing thymocytes and non-lymphoid cells to long-lasting gonadal deprivation

Promene u modelu diferentovanja timocita u mužjaka pacova sa godinama

In order to elucidate the features of putative age-related changes in the intrathymic T cell maturation sequence, the thymus weight, thymocyte yield and relative proportions of thymocyte subsets were analyzed at distinct maturational stages (delineated by analysis of the expression of CD4/CD8 coreceptor molecules and TCR molecular complex) in male AO rats, from the peripubertal period (1 month of age) until 10 months of age. In 2-month-old rats both the thymus weight and lymphoid content were greater than in 1-month-old rats. The values of both parameters in 4-month-old rats were reduced to the corresponding values in 1-month-old rats. These levels were sustained until 10 months of age. However, the thymocyte composition was subjected to substantial changes during the whole period studied, probably due to alterations in action of both extrinsic and intrinsic factors which influence the ability of the thymus to support T cell maturation and/or feedback regulatory action of intrathymic CD4+ T cells on thymocytopoiesis (between the age of 7 and 10 months).U cilju definisanja karakteristika procesa sazrevanja T ćelija u timusu u funkciji uzrasta, u mužjaka pacova AO soja, različitog uzrasta (od 1 meseca do 10 meseci) posmatrane su: težina timusa, ukupan broj timocita, kao i relativna zastupljenost pojedinih timocitnih subpopulacija (koje su razdvojene na osnovu ekspresije CD4/8 koreceptornih molekula i antigena TCRαβ molekulskog kompleksa). U životinja uzrasta 2 meseca i težina timusa i ukupan broj timocita bili su značajno veći nego u životinja uzrasta od mesec dana. Međutim, u životinja uzrasta od 4 meseca vrednost ovih parametra nije se razlikovala od vrednosti odgovarajućih parametara sa mesec dana. Vrednosti oba ova parametra nisu se bitno menjale od 4. do 10. meseca života. Sa druge strane, naši rezultati su pokazali značajne promene u relativnom odnosu timocitnih subpopulacija u ispitivanom periodu. Ove promene se mogu objasniti promenama u delovanju, kako spoljašnjih tako i unutrašnjih, faktora koji modulišu sposobnost timusne mikrosredine da podrži proces sazrevanja T ćelija i/ili utiču na efikasnost regulatornog delovanja intratimusnih CD4+ T ćelija, mehanizmom negativne povratne sprege, na proces sazrevanja T ćelija u timusu

Modulacija eksperimentalnog autoimunog encefalomijelitisa (EAE) DA pacova levamizolom

We investigated the influence of an antiparasitic drug, levamisole (2,3,5,6 - tetrahydro - 6- phenyl-imidazo (2,1 - b) thiazole -hydrochloride) with potent immunomodulatory properties on the course and development of experimental autoimmune encepha-lomyelitis (EAE). EAE was induced in female Dark Agouti (DA) rats aged two months by immunization with guinea pig spinal cord in complete Freunds adjuvant. Following immunization animals were subcutaneously treated every other day with 2.2 mg/kg levamisole. The course, development and characteristics of this autoimmune process were monitored as indirect indicators of immune system activity. Our results indicate that in EAE levamisole exerts immunosuppressive effects when administered every other day from the moment of immunization until the end of the disease. This application regime and dose postponed the onset of the first clinical signs, shortened the duration of the disease, abrogated the severity of clinical symptoms and accelerated the recovery of sick animals. In the period of induction and during EAE, levamisole also decreased the severity of changes in the cerebral perivascular spaces. In the peripheral blood of levamisole treated animals with induced EAE, a significant increase of CD4-CD8+ T cells was demonstrated. Furthermore, all rats with induced EAE had decreased numbers of CD4+CD8- T cells in their blood. These changes were in correlation with clinical signs of EAE.U ovom radu su prikazani rezultati ispitivanja anthelmintika levamizola (2,3,5,6 tetrahidro - 6 - fenil - imidazo (2,1 - b) tiazol hidrohlorida) sa snažnim imunomodulatornim svojstvima na tok i razvoj eksperimentalnog autoimunog encefalomijelitisa (EAE). EAE je indukovan imunizacijom ženki pacova soja DA (Dark Agouti) starih dva meseca pomoću homogenata kičmene moždine zamorčeta u kompletnom Freundovom adjuvansu. Posle imunizacije, životinje su tretirane subkutanim injekcijama levamizola (2.2 mg/kg) svaki drugi dan a praćeni su tok, razvoj i karakteristike ovog autoimunog oboljenja kao indirektni indikatori aktivnosti imunološkog sistema. Postignuti rezultati ukazuju dalevamizol ispoljava imunosupresivno delovanje u modelu EAE ako se aplikuje svaki drugi dan od momenta imunizacije do kraja bolesti. Primenjena doza i režim aplikacije odložili su momenat pojavljivanja prvih kliničkih simptoma, skratili trajanje bolesti, ublažili ispoljavanje simptoma i ubrzali oporavak bolesnih životinja. U periodu indukcije i tokom EAE-a levamizol je smanjio stepen promena u cerebralnim perivaskularnim prostorima. U ženskoj krvi ženki pacova sa indukovanim EAE i tretiranim levamizolom uočeno je značajno povećanje broja CD4-CD8+ T ćelija. Osim toga, u obe imunizovane grupe životinja zapaženo je smanjenje broja CD4+CD8- ćelija. Ove promene su bile u skladu sa kliničkom slikom bolesti

Diferentovanje timocita u organ kulturi timusa odraslih pacova

To investigate the differences between thymocytes development in vivo and in vitro, thymus lobe fragments from 12-weeks old male Albino Oxford rats were cultivated over a 7-days period. In the controls and cultivated thymic lobes fragments were evaluated and the viability, apoptosis and cell cycle of thymocytes, as well as the histological characteristics of thymic tissue. Additionally, we analyzed the expression of CD4, CD8 and TCRαβ on thymocytes by flow cytometry. The obtained results showed that thymus cellularity decreased during cultivated time due to expanded apoptosis, decreased proliferation and the absence of progenitors reseeding thymus. The relative proportion of thymocyte subsets in the first 24 hours of culture remained similar as in the control. However, cultivation for 3 and 7 days modulated the relative proportions between thymoctye subsets. The percentage of DP TCRαβlow increased, DP TCRαβhi subset remained unchanged, both SP TCRαβhi subsets decreased while the same mature SP phenotype dominated in culture media. These results demonstrate that cultivated thymic fragments retain the capacity for T cell development, although cultivation modulates this process.Sa namerom da ispitamo razlike između in vivo i in vitro sazrevanja timocita gajili smo fragmente lobusa timusa poreklom od mužjaka Albino Oksford pacova, starih dvanaest nedelja, u vremenskom periodu od sedam dana. Nakon kultivacije određivani su vijabilnost, apoptoza i ćelijski ciklus timocita, kao i histološke osobine timusnog tkiva. Takođe je analizirano ispoljavanje markera diferentovanja CD4, CD8 and TCRαβ na površini timocita metodom tečne citofluorometrije. Dobijeni rezultati su ukazali da sedmodnevna kultivacija dovodi do smanjenja broja ćelija u timusu usled povećane apoptoze, smanjene proliferacije i odsustva ulaska progenitora timocita. Tokom prvih 24 sata kultivacije ne dolazi do promena u odnosima timocitnih populacija. Međutim, duže vreme kultivacije - 3 i 7 dana moduliše relativne odnose između timocitnih subpopulacija - povećava se procenat DP TCRαβlow, procenat DP TCRαβhi timocita ostaje nepromenjen, dok su procenti ćelija oba subseta SP TCRαβhi smanjeni, mada je prisustvo pomenutih SP subsetova dominantno u medijumu za kultivaciju. Navedeni rezultati pokazuju da kultivisani fragmenti timusnog tkiva zadržavaju sposobnost da podrže sazrevanje timocita u jednostruko pozitivne T ćelije, mada je diferentovanje timocita donekle modulisano kultivacijom

Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development

The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of “inflammescent” cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy

Peripubertetna ovarijektomija obezbeđuje dugotrajno odlaganje starenjem uslovljenog smanjenja celularnosti timusa i produkcije T limfocita

The present study was undertaken to reassess the recently challenged role of ovarian hormones in age-associated thymic involution. For this purpose, in eleven-month-old peripubertally ovariectomized (Ox) rats we analyzed: i) thymic weight and cellularity, ii) size of CD4+CD8+ double-positive (DP) thymocyte population, which is believed to correlate to the thymic capacity to export mature T cells, iii) number of recent thymic emigrants (RTEs), and iv) number of peripheral blood CD4+ and CD8+ lymphocytes. It was found that both thymic weight and cellularity were greater in Ox than in control rats. In addition, in Ox rats the numbers of DP thymocytes and both CD4+ and CD8+ RTEs, were significantly greater than in controls, indicating a more efficient generation of T cells in these rats. Furthermore, these findings, coupled with data indicating that the number of neither CD4+ nor CD8+ peripheral blood lymphocytes was affected by ovariectomy, most likely, suggest a reduced homeostatic proliferation of memory cells in Ox rats, i.e. broadening of TCR peripheral repertoire without changes in the overall number of T cells leading to a more efficient response to newly encountered antigens. The results indicate that the ovarian steroid deprivation from early peripubertal period leads to a long lasting postponement/alleviation of age-associated decline in T-cell mediated immune response.Ova istraživanja su preduzeta sa ciljem da se preispita uloga gonadnih hormona u involuciji timusa, koja je nedavno dovedena u pitanje. U tom cilju je kod 11 meseci starih ženki pacova, koje su ovarijektomisane (Ox) u peripubertetnom uzrastu, analizirana: i) težina i celularnost timusa, ii) broj CD4+CD8+ dvostruko pozitivnih (DP) timocita, za koji se smatra da odražavaju sposobnost organa da generiše zrele T limfocite, iii) broj neposrednih emigranata iz timusa (RTE) i iv) ukupan broj CD4+ i CD8+ limfocita u perifernoj krvi. Dokazano je da su težina i celularnost timusa bile značajno veće u Ox životinja. Kod ovih životinja je nađen i povećan broj DP timocita, kao i CD4+ i CD8+ RTE, što ukazuje na efikasniju produkciju T ćelija u njihovom timusu. Ovaj nalaz, u kontekstu nepromenjenog broja CD4+ i CD8+ ćelija u perifernoj krvi, takođe sugeriše smanjenu homeostatsku proliferaciju memorijskih ćelija, odnosno ukazuje na kvalitativne promene u perifernom T ćelijskom repertoaru (koje obezbeđuju efikasniji odgovor na nove antigene) bez kvantitativnih promena. U celini, rezultati ukazuju da u odsustvu hormona ovarijuma počevši od ranog peripubertetnog uzrasta dolazi do značajnog odlaganja/ublažavanja involucije timusa i posledičnih promena na periferiji

Strain differences in thymic atrophy in rats immunized for EAE correlate with the clinical outcome of immunization

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCR alpha beta-mediated signaling and activation thresholds, on CD4+CD8+ TCR alpha beta(lo/hi) thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCR alpha beta(hi) thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16(INK4a) accumulation). The higher circulating level of TNF-alpha in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with their AO counterparts. In conclusion, the study indicated that strain differences in immunization-induced changes in thymopoiesis and peripheral CD4+CD28- T-cell generation could contribute to rat strain-specific clinical outcomes of immunization for EAE

Fenotipske promene izazvane imunizacijom encefalitogenom menjaju funkcije peritonealnih makrofaga u dva soja pacova različite osetljivosti prema indukciji eksperimentalnog autoimunskog encefalomijelitisa (EAE).

We have investigated the phenotype of peritoneal cells and the functions of peritoneal macrophages obtained from experimental autoimmune encephalomyelitis (EAE)-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rat strains on days 1, 3 and 7 post immunization with encephalitogen. Resident peritoneal cells from immunized and non-immunized rats of both strains were subjected to flow cytometric analyzes and after adherence were tested for zymosan phagocytosis, hydrogen peroxide (H2O2) and nitric oxide (NO) production. In non-immunized rats, macrophages from the DA rat strain phagocytosed more zymosan but produced less H2O2 than cells from the AO strain, while both strains produced comparable amounts of NO. Immunization increased phagocytosis in DA rats' cells, but decreased both phagocytosis and H2O2 production in cells from AO rats. Overall higher phagocyte ability in DA rats was associated with a significantly larger population of ED1+ cells (macrophages and dendritic cells), in contrast to a more pronounced expression of ED2 antigen (resident macrophages) on cells from AO rats. Immunization also increased the expression of CD11b molecule on non-resident ED2-macrophages of DA, but not of AO rats. The early and subtle phenotype changes in peritoneal cells of both rat strains might mirror the mechanism contributing to their different sensitivity to the induction of autoimmunity.Ispitivan je fenotip peritonealnih ćelija, kao i funkcije peritonealnih makrofaga, izolovanih od pacova Dark Agouti (DA) soja osetljivog na indukciju eksperimentalnog autoimunskog encefalomijelitisa (EAE) i pacova Albino Oxford (AO) soja koji je rezistentan prema EAE-u, 1, 3. i 7. dana nakon imunizacije encefalitogenom. Rezidentne peritonealne ćelije su ispitivane metodom protočne citofluorometrije, a zatim je nakon adherence testirana njihova sposobnost fagocitoze čestica zimozana i kapacitet produkcije vodonik peroksida (H2O2) i azot monoksida (NO). U neimunizovanih pacova makrofage DA soja su intenzivnije fagocitovale čestice zimozana i imale nižu sposobnost produkcije H2O2 nego ćelije pacova AO soja, ali nije bilo sojnih razlika u sposobnosti produkcije NO. Imunizacija je dovela do povećanja fagocitne sposobnosti makrofaga DA pacova, ali i do smanjenja fagocitoze i produkcije H2O2 makrofaga pacova AO soja. Generalno veću sposobnost fagocitoze u DA pacova prati i značajno veća zastupljenost ED1+ ćelija (koje čine uglavnom makrofage i dendritične ćelije) nasuprot većoj zastupljenosti ED2 antigena (marker rezidentnih makrofaga) na ćelijama pacova AO soja. Imunizacija encefalitogenom je takođe dovela do povećanja ekspresije CD11b molekula na nerezidentnim ED2- ćelijama pacova DA, ali ne i AO soja. Rane i diskretne fenotipske promene na peritonealnim ćelijama pacova oba soja verovatno odslikavaju mehanizme koji doprinose njhovoj različitoj osetljivosti prema indukciji autoimunskih oboljenja

Strain differences and the role for HSP47 and HSP70 in adjuvant arthritis in rats

Because of high sequence homology between microbial and endogenous heat shock proteins (HSP), immunological cross-reactivity to microbial HSP has been suggested as a possible cause of the development of autoimmune diseases, such as rheumatoid arthritis. The present study aimed to determine a potential role of HSP47, a molecular chaperone involved in the synthesis and assembly of collagen molecules, and microbial HSP71 (mHSP71) in adjuvant arthritis (AA) in two rat strains: Dark Agouti (DA), susceptible to AA induction and Albino Oxford (AO), which is resistant to AA induction. Immunization with complete Freund's adjuvant (CFA) induced an increased expression of HSP47 in joints of DA rats, which exhibited severe clinical signs of AA at the time of disease peak, while this protein was not detectable in joints of AO rats. In contrast, no strain differences in HSP72 (rat analogue of mHSP71) expressions in joints were observed. The increased levels of anti-HSP47 antibodies were detected in sera of DA rats during the AA peak, while the immunization with CFA increased levels of anti-mHSP71 antibodies in sera of AO rats. HSP47 and mHSP71 reduced proliferation of draining inguinal lymph node cells (LNC) in resistant AO rat strain, leading to a hypothesis that both HSP participated in AA control. Finally, mHSP71 potentiated the apoptotic response of LNC in susceptible DA rat strain. In conclusion, our findings indicate involvement of HSP47 in the development of AA in the rat, and point out to the regulatory role for both HSP47 and mHSP71

Peripubertal orchidectomy transitorily affects age-associated thymic involution in rats

The role of gonadal hormones in induction and, particularly, maintenance/ progression of rat thymic involution, which normally starts around puberty, was reassessed by examining the effects of peripubertal orchidectomy on thymic weight and morphometric parameters at different times up to the age of 10 months. Up to 6 months post-castration both thymic weight and cellularity in orchidectomized ( Cx) rats were greater than in age-matched control rats, sham Cx ( Sx). The increase in thymic cellularity reflected an increase in thymocyte proliferation rate ( the proportion of proliferating cells was 18.6 +/- 0.7% in 2-month-old Cx ( N = 5) vs 13.4 +/- 0.3% ( N = 5) in age-matched Sx rats) followed by reduced sensitivity to apoptotic signals ( apoptotic thymocytes were 9.8 +/- 0.9% in 2-month-old Cx ( N = 5) vs 15.5 +/- 0.3% ( N = 5) age-matched Sx rats). However, 9 months post-orchidectomy, neither thymic weight and cellularity nor any of the morphometric parameters analyzed differed between Cx and control rats. The reduction of thymic cellularity in Cx rats to control values may be related to increased sensitivity of their thymocytes to apoptotic signals in culture ( 72.6 +/- 1.2% in 10-month-old vs 9.8 +/- 0.9% in 2-month-old Cx rats) followed by reduced responsiveness to proliferative stimuli ( 14.1 +/- 0.2% in 10-month-old vs 18.6 +/- 0.7% in 2-month-old Cx rats). Thus, the study indicates that the effects of peripubertal orchidectomy on thymic weight and cellularity, as well as on the main morphometric indices, are long-lasting but not permanent, i.e., that removal of the testes can only postpone but not prevent age-related organ atrophy and consequently functional deterioration of the immune system