ainio-Pöllänen S

Role of transforming growth factor β in testicular immunosuppression

Abstract The potential role of transforming growth factor β (TGFβ) in the regulation of the immunological milieu of the testis was investigated. Antibodies neutralizing TGFβ reversed the previously observed suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract. Recombinant TGF β 1 dose-dependently inhibited testicular interleukin-1-like factor-driven proliferation of murine thymocytes and ConA-stimulated rat peripheral blood mononuclear cells. Extracts of seminiferous tubules contained a M r ∼25 K TGFβ-like growth inhibitor of the CLL-64 mink lung epithelial cell line. The present findings suggest an important role for TGFβ in testicular immunosuppression.

CD28–CD80/CD86 Interactions in testicular immunoregulation

The expression of two accessory molecules on antigen-presenting cells (APC), the CD80/ B7-1 and CD86/B7-2 antigens, was studied in the testis of normal and non-obese diabetic (NOD) mice. In addition, the effect of CD28 stimulation on suppression of lymphocytes by testicular products was investigated. The testes of 4-week old NOD mice or normal BALB/c mice and the testis of 17-21-week old BALB/c mice contained no CD80 or CD86 expressing cells. In contrast, CD80+ and CD86+ cells were present in the testis of 14-22-week old NOD mice. The CD80+ cells and most of the CD86+ cells were CD11b/CD18 negative. There were some CD11b/CD18+ cells that expressed CD86 weakly. The CD80+ and CD86+ cells were often located adjacent to the vessel walls where a leukocyte not expressing CD80 or CD86 had attached to the endothelium. Some CD80+ and CD86+ cells were present among the interstitial cells. The CD80 and CD86 antigens could not be observed in the same cells as judged from stainings in parallel sections. Stimulation of ConA- or anti-CD3 epsilon-primed peripheral blood or spleen lymphocytes with anti-CD28 was able significantly to antagonize the growth-inhibitory effect of the M(r) > 5 K fraction of testis extracts, but could not abolish it with increasing concentrations of testis extract. The results suggest that T lymphocytes can not be activated locally in the testis of BALB/c and young NOD mice because of the absence of the necessary CD28 ligands, CD80 and CD86, from the APCs and because of the suppression of T lymphocytes by the testicular products. In the testis of older diabetic NOD mice lymphocyte activation may occur because the testes of these mice contain CD80+; CD11b/CD18-, CD86+; CD11b/CD18+ and CD86+; CD11b/CD18- cells and therefore, CD28 co-stimulation, which can antagonize the suppressive effect of testis extract, may occur. The possibilities for clonal anergy in testicular immunoregulation are discussed.

CD106 (VCAM-1) in testicular immunoregulation

The expression of CD18, CD49d/CD29, CD44, CD54 and CD106 was studied in the testis of normal mice at various ages, in the cryptorchid testis, in the testis of estrogen-treated mice and in the testis of non-obese diabetic (NOD) mice, using immunocytochemistry to see which of these lymphocyte and endothelial adhesion proteins may be involved in lymphocyte regulation in the testis. CD18-, CD49d/CD29-, CD44- and CD54-expressing cells were not found in the normal > 10-week-old BALB/c mouse testis. Leydig cells expressed CD106 strongly at this age. In contrast to the > 10-week-old testis, only very few interstitial cells of the 2-week-old normal mice expressed CD106. The expression of CD106 increased gradually with age so that at 6 weeks of age the expression of CD106 was moderate in the interstitial tissue. In the experimentally abdominal testis, CD106 was expressed in the interstitial tissue as strongly as in the contralateral scrotal testis. CD44- and CD18-expressing cells were occasionally present in the interstitial tissue of the abdominal testis, but not in the contralateral scrotal testis. CD54 was present in the epithelium of the ductuli efferentes. In the testis of the estrogen-treated mice, CD106 was expressed in the interstitial tissue as strongly as in the normal mice. Occasional CD44- and CD18-expressing cells were found in the testicular capsule. In the testis of adult NOD mice, CD106 was present in the interstitial tissue, but none of the other studied proteins. Immunoblotting of CD106 from the adult testis under reducing conditions demonstrated a single broad band with a M(r) of 51-65 kDa. This is a novel isoform of CD106. In a modified Stamper-Woodruff assay, lymphocytes bound to the testicular interstitial tissue. In co-incubations of native Leydig cells and lymphocytes, anti-CD106 antibodies prevented formation of Leydig cell-lymphocyte rosettes more than isotype-matched irrelevant control antibodies, suggesting that Leydig cell lymphocyte binding occurs through CD106-CD49d interactions. In lymphocyte cultures in the presence of anti-CD3, anti-CD28, the M(r) > 5 K fraction of testis extract (containing CD106 as shown by immunoblotting) and anti-CD106 or control antibody, anti-CD106 did not consistently affect T cell 3H-TdR incorporation. The present results suggest that CD106 expressed by the Leydig cells may act as an adhesion-promoting molecule or a co-stimulatory factor for T cells migrating to the testis.

Ontogeny of immunosuppressive activity, MHC antigens and leukocytes in the rat testis

The levels of immunosuppressive activity and the presence of MHC antigens and leukocytes were studied in the immature and the sexually mature rat testis. The immunosuppressive activities were measured from high-molecular weight (greater than 5 kDa) fractions of testis extracts using the protectin bioassay. The presence of MHC antigens and leucocytes was studied using the indirect immunoperoxidase method. In the immature rats, clusters of class I MHC antigen positive cells and a few cells expressing class II MHC antigen were present in the testicular interstitium. In the sexually mature rats, all the cells were MHC I+, and MHC II+ cells were numerous in the testicular interstitium. The seminiferous epithelium was MHC-negative in both the immature and the sexually mature testis. W3/25+ leukocytes were present in the interstitium and the tubular wall in both the immature and the sexually mature rat testis, but not in the seminiferous epithelium at any age. At 20-30 days of age, the testicular extracts were neutral or slightly stimulated 3H-TdR incorporation into peripheral blood lymphocytes, but at 44-60 days of age they inhibited lymphocyte proliferation significantly. In gel filtration, a peak of immunosuppressive activity was observed at approximately 400 kDa (protectin A) in both 20- and 60-days-old rat testes. A smaller peak was present at approximately 200 kDa in both age groups. This study shows that the testicular immunoregulatory microenvironment is different in the immature and the sexually mature rats. This may be important in such age-dependent human diseases as mumps orchitis and the testicular relapses of acute lymphoblastic leukemia.

The role of Fas ligand in the development of insulitis in nonobese diabetic mice

The role of Fas ligand-induced lymphocyte apoptosis in the development of insulitis was studied in nonobese diabetic (NOD) mice. Fas ligand with a molecular weight of 45 kD appeared in the pancreas of NOD mice during the onset of insulitis as demonstrated by western blot analysis. In situ DNA end-labeling (ISEL) of the pancreases of NOD mice demonstrated positive cells in the islets of Langerhans, with an age-dependent increase in the density and frequency of animals with islets containing ISEL-positive cells. In the pancreatic islets of BALB/c mice, no ISEL-positive cells were observed in any of the age groups studied. In ultrastructural analysis degenerating cells with condensed or fragmented nuclei and plasma membranes detached from neighboring cells were observed both in and around the islets. In some cases, these cells were being phagocytosed by the neighboring islet cells. Degenerating cells with characteristics of lymphocytes were seen in contact with healthy lymphocytes around the islets. Neutralizing Fas ligand antibodies affected 3H-thymidine incorporation of CD3+CD28+ pancreatic lymphocytes from 12- to 30-week-old NOD mice in one of three cultures. There was no difference in the effect of neutralizing Fas ligand antibodies between pancreatic and blood lymphocytes. The present results on an increase in density of apoptotic cells in pancreatic islets of NOD mice simultaneously with the onset of insulitis and appearance of Fas ligand suggest that the pancreas infiltrating lymphocytes may be destroyed by Fas ligand-induced apoptosis.

Immunosuppressive activity in the rat seminiferous tubules

Rat seminiferous tubule segments in defined stages of the epithelial cycle were isolated by transillumination-assisted microdissection. The segments were cultured together with ConA-stimulated peripheral blood lymphocytes (PBL) and incorporation of 3H-labelled thymidine was measured. Tubule segments in stages II-VIII of the seminiferous epithelial cycle inhibited PBL proliferation significantly more than stages IX-I. Inhibition was lowest in stages IX-XII and increased progressively to reach a maximum in stages II-VIII. In a more detailed analysis, tubules in stages V and VI inhibited PBL proliferation significantly less than stage II tubules. No significant difference was observed between stages II and VII. The immunosuppressive activity had molecular weights of approximately 25 kDa and approximately 65 kDa in stage II-VIII seminiferous tubules. In stage II-VI seminiferous tubules activity was present also at approximately 10 kDa. The results suggest that the seminiferous tubules produce high-molecular weight immunosuppressive activity in a stage-dependent way. In addition to its contribution to the immunologically privileged milieu of the testis this activity may also be involved in the physiological regulation of DNA synthesis in the seminiferous epithelium.

Cytokine Balance and Lipid Antigen Presentation in the NOD Mouse Pancreas During Development of Insulitis

The role of cytokine balance and lipid antigen presentation in the development of diabetes was studied using immunohistochemistry of cytokines in the pancreas of nonobese diabetic mice (NOD) and BALB/c mice at various ages. In both the NOD and BALB/c mice, interleukin 10 (IL-10) was expressed in the islets. IL-10 was also present in the epithelial cells of the exocrine tissue in both strains. In the NOD mice, IL-10 disappeared from both the islets and the exocrine tissue at 16 weeks of age. At this age, IL-10 was still present in the islets and exocrine tissue of the BALB/c pancreata. IL-10 was not present in the pancreata of diabetic NOD mice. IL-6 first appeared in the pancreas at 10 weeks of age and disappeared at the age of 16 weeks in both NOD and BALB/c mice. It was present in the endothelial cells. Neither the pancreata of normal BALB/c mice nor NOD mice at 2–16 weeks of age contained tumor necrosis factor &agr; (TNF-&agr;), interferon &ggr; (IFN-&ggr;), IL-4, or IL-12. At 8 weeks of age, a few IL-2+ cells were found in the pancreas of one of three NOD mice. CD1d was already present in both strains at 2 weeks of age but disappeared from the NOD mice at 16 weeks of age. CD1d localized to walls of tubular structures probably representing collecting tubules. These results suggest that in the NOD mice the disappearance of the TH0, TH1, and TH2 responses inhibiting IL-10 from the islets at the age of 16 weeks may trigger the final stage of the immune response leading to overt diabetes. The simultaneous disappearance of CD1d suggests that activation of immune responses against lipid antigens does not play a role in this stage of the disease.

The Role of Lipid Antigen Presentation, Cytokine Balance, and Major Histocompatibility Complex in a Novel Murine Model of Adoptive Transfer of Insulitis

After adoptive transfer of insulitis from nonobese diabetic (NOD) mice, leukocytes accumulate in the pancreas of SCID/SCID and NOD/SCID mice. These cells express classical antigen-presenting molecules and costimulators of T-cell activation and adhesion molecules involved in homing. The aim of the present study was to study the expression of cytokines involved in regulation of the TH1/TH2 balance by these cells, the role of lipid antigen presentation in the local immune system activation in the pancreas during onset of insulitis, and the role of major histocompatibility complex in this process. Splenocytes from NOD and BALB/c mice were injected intraperitoneally to SCID/SCID and NOD/SCID mice. Sections from the pancreata of these injected mice were stained for cytokines (tumor necrosis factor &agr; [TNF-&agr;], interferon gamma [IFN-&ggr;], CD1d, interleukin 2 [IL-2], IL-4, IL-6, IL-10, and IL-12). Some SCID/SCID and NOD/SCID mice injected with NOD splenocytes developed a severe disease. IL-10 was expressed in almost all the animals: in exocrine pancreas, large groups of infiltrating lymphocytes, endothelia of blood vessels, pancreatic islets, and interstitial tissue. CD1d was found in most of the mice: in the endothelia of collecting ducts and blood vessels of the pancreas, lymphocytic infiltrates, interstitial tissue, septae, islets, and a pancreatic lymph node. TNF-&agr; was expressed notably more often in the pancreata of NOD/SCID than SCID/SCID mice. It was found between pancreatic lobules, in the epithelia of collecting ducts, endothelia of blood vessels, islets, capillaries, infiltrates, and septae. IL-6 was expressed more in the SCID/SCID than in the NOD/SCID mice. It was seen in infiltrates, walls of blood vessels, around islets, and in connective tissue. IFN-&ggr; was found only in the pancreata of SCID/SCID and NOD/SCID mice injected with NOD splenocytes. The expression of IL-2 and IL-12 was very scarce. IL-4 was not expressed at all. The present study clearly shows that antigen presentation has a role in the development of autoimmune diseases after adoptive transfer of splenocytes from diseased mice to intact ones and that IL-10 may have a central role in the control of the disease process.

Appearance of Cells Expressing CD80 and CD86 Costimulatory Antigens in the Pancreas of Nonobese Diabetic Mice

The immunopathogenetic mechanisms that lead to type I diabetes in the nonobese diabetic mouse are poorly defined. The immunoregulatory effects of the costimulatory CD80/B7-1 and CD86/B7-2 antigens expressed by the antigen-presenting cells may be crucial for the development of immune responses and their absence may lead to clonal anergy, which is important in the prevention of autoimmune reactivity. To evaluate the role of the CD80 and CD86 antigens in the development of insulin-dependent diabetes mellitus, expression of these antigens in the pancreas of normal and nonobese diabetic mice was studied. The pancreata of normal BALB/c mice and young nonobese diabetic mice contained no CD80- or CD86-positive cells. CD80- and CD86-positive cells appeared in the pancreas of nonobese diabetic mice by 6 weeks of age. Double immunostaining at the age of 12 weeks showed that CD80-positive cells accumulated around the islets of Langerhans but no insulin-containing cells expressed the costimulatory molecules. The morphology of the CD80- and CD86-positive cells was heterogeneous. Many were Mac-1 integrin (CD11b/CD18) positive, suggesting that macrophages expressing the costimulatory antigens are present in the pancreas of all nonobese diabetic mice by 12 weeks of age and that CD80- and CD86-expressing macrophages may be involved in the local regulation of antiislet immune responses.

The role of CD8+ cells, cell degeneration, and Fas ligand in insulitis after intraperitoneal transfer of NOD splenocytes

Cells expressing CD4, CD8, CD18, CD49d/CD29, CD44, CD54, CD80, CD86, CD106, CD11b/CD18 or DNA breaks were stained in the pancreases of female or male scid/ scid mice after adoptive transfer of lymphocytes from older than 12-week female nonobese diabetic (NOD) or Balb/c mice. After intraperitoneal adoptive transfer of NOD splenocytes to female severe combined immunodeficiency (scid)/scid mice, in situ end labeling (ISEL)+ as well as CD80+ and CD86+ cell infiltrates appeared first in the blood vessel walls and pancreatic interstitial tissue at 2-3 weeks after transfer. CD4+, CD8+, CD18+, CD44+, CD54+, and CD106+ cells then encircled and invaded some islets of the scid/scid mice 2 to 7 weeks after transfer. Cells expressing these surface components, except CD8, were present also in the Balb/c mice, but as individual cells, not as infiltrates. CD8+ cells were observed in the pancreases of all NOD splenocyte-injected scid mice at 2, 3, 4, 6, and 7 weeks after transfer, but in none of the Balb/c splenocyte-injected scid mice. Some scid/scid mice injected with NOD splenocytes also developed severe noninfectious diarrhea and cachexia 4 weeks after transfer. ISEL+ cells were observed in the pancreases of NOD splenocyte-injected female scid mice at all times after transfer, especially in the blood vessel walls and in the islets. Fas ligand was not present in Western blotting. It is proposed that apoptosis commonly occurs in islet-infiltrating lymphocytes and that in the scid/scid adoptive-transfer model, the first islet-infiltrating cells are destroyed by programmed cell death independent of Fas ligand. Further, CD8+ T lymphocytes inevitably play a central role in intraperitoneal adoptive transfer of insulitis.