Brian E. Fuller

Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression.

Mechanisms suppressive to induction of murine experimental autoimmune thyroiditis (EAT) can be activated by pretreatment with tolerogenic doses of mouse thyroglobulin (MTg) or prior TSH infusion to raise circulatory MTg levels. MTg-activated suppressor T cells (Ts), shown earlier to be Thy-1+ and probably I-J+, were further characterized by in vivo administration of paired rat monoclonal antibodies to distinct epitopes on the L3T4 or Lyt-2 molecule, either on the day of, or subsequent to, initiation of the tolerogenic regimes. The cells required at the time of MTg pretreatment were L3T4+, Lyt-2- and low anti-L3T4 doses had no effect on their activation. The cells that mediated the strong MTg-induced resistance following pretreatment were also L3T4+; their suppressor function could only be abrogated by depletion of L3T4+, but not Lyt-2+, cells. Injection of cyclophosphamide (20-100 mg/kg) either prior to EAT induction or after Ts activation did not affect the severity of disease. Similarly, the suppressor state evoked by TSH infusion could only be abrogated by anti-L3T4 treatment. These findings indicate that both MTg-activated and TSH-induced suppression are mediated by L3T4+ cells. We hypothesize that MTg-specific Ts are present in normal, EAT-susceptible mice in low numbers to contribute to the maintenance of self-tolerance and that they are stimulated by increased levels of circulatory MTg to expand/differentiate and mediate the marked resistance to EAT induction.

Role of mouse and human class II transgenes in susceptibility to and protection against mouse autoimmune nhyroiditis

Abstract Mouse experimental autoimmune thyroiditis (EAT), a model for Hashimoto’s thyroiditis, is induced by immunizing with mouse thyroglobulin (MTg). To study the extent of H2A involvement in EAT, we introduced AaAb genes from susceptible k mice into resistant or intermediately susceptible strains which do not express H2E molecules. Thyroiditis was severe in resistant B10.M (H2f) mice carrying the double transgene AakAbk. Likewise, thyroid infiltration was significantly extended in intermediate B10.Q (H2q) mice with the same transgene. To examine the effect of H2E molecules in the presence of H2A-mediated susceptibility, we introduced an Eaktransgene into E– B10.S mice to express the Eβs molecule and observed significant reduction in EAT severity in B10.S(E+) mice. On the other hand, the presence of an Ebd transgene in B10.RQB3 (H2Aq) mice resulting in the expression of H2Eβd molecules did not alter EAT susceptibility, suggesting a role for Eb gene polymorphism in protection against EAT. We have shown recently that the HLA-DRB1*0301 (DR3) transgene conferred EAT susceptibility to B10.M as well as class II-negative B10.Ab0 mice. However, we report here that the HLA-DQB1*0601 (DQ6b) transgene in B10.M or HLA-DQA1*0301/DQB1*0302 (DQ8) transgene in class II-negative Ab0 mice did not. These studies show the differential effects of class II molecules on EAT induction. Susceptibility can be determined when class II molecules from a single locus, H2A or HLA-DQ, are examined in transgenic mice, but the overall effect may depend upon the presence of both class II molecules H2A and H2E in mice and HLA-DQ and HLA-DR in humans.

Depletion of CD4+ and CD8+ cells eliminates immunologic memory of thyroiditogenicity in murine experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT) develops in genetically susceptible mice after immunization with mouse thyroglobulin (MTg), and is mediated by T cells, both CD4+ and CD8+, infiltrating the thyroid. Previous work showed that depletion of CD4+, but not CD8+, cells with rat monoclonal antibodies (mAbs) interfered with EAT induction. To test if concomitant CD4+ cell depletion and immunization led to EAT resistance, mice were reimmunized at an interval of 15 or 43 days after injection of CD4 mAbs. No resistance had been established; disease severity and anti-MTg titers were comparable to mice with primary immunization. Previous work also showed that treatment during advancing EAT with only CD4 mAbs on days 21, 25 led to long-lasting, reduced severity in EAT, whereas administration of CD8 mAbs alone reduced the smaller CD8+ subset only. However, therapy with both mAbs was most efficacious; > 50% of thyroids were purged of all cellular infiltrate after only two doses. Moreover, T cells emerging subsequent to depletion were not retained in the thyroid, despite ongoing antibody production. To test if nondepleting CD4 and CD8 mAbs were similarly effective for therapy, mAbs of the IgG2a isotype were administered during advancing EAT. No effect on thyroidal infiltration was observed, indicating that modulation of the CD4 and CD8 antigen without depletion was insufficient for efficacious therapy. To determine if combined therapy with depleting mAbs reestablished self tolerance, treated mice were reimmunized on days 70, 77, when T cell recovery was nearly complete. Thyroiditis was comparable to controls given primary immunization, despite high antibody levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the d.c. photoelectric signal from model bacteriorhodopsin membranes: d.c. photoconductivity determination by the null current method and the effect of proton ionophores

Abstract On illumination by continuous light, a reconstituted bacteriorhodopsin membrane exhibits a stationary photocurrent under short-circuit conditions. It has been widely reported that this photocurrent is linearly dependent on the applied transmembrane potential, and that the photocurrent reverses its polarity at a critical potential. It is also well known that the stationary photosignal of a bacteriorhodopsin membrane is linearly dependent on the light intensity and eventually reaches saturation. In this paper, the null current method (F.T. Hong and D. Mauzerall, Biochim. Biophys. Acta, 275 (1972) 479) is applied to decompose the photocurrent into a photovoltaic part (photoemf) and a photoconductive part (photoconductance). It is found that the photoconductance is zero in the dark, and is activated by illumination to reach a fixed magnitude which is independent of a further increase in the light intensity (“step-function” photoswitching). Furthermore, the photoconductance is ohmic (i.e. independent of the applied potential). The linear voltage dependence of the photocurrent can be explained in terms of the photoswitching by assuming that the light-activated proton conductance channel is also available for a transmembrane potential to drive a proton current through in either direction. With this assumption, the photoemf is shown to be voltage independent. The photoemf is initially linearly light dependent at low light intensities, but eventually reaches a saturation level. We confirm the reported enhancement effect of the proton ionophores carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), which is caused by increases in both the photoconductance and the ionic conductance. The action of the Cl− ionophore, nystatin, is quite different. Nystatin inhibits the photocurrent and increases the ionic conductance, but does not affect the photoconductance. The enhancement effect of proton ionophores cannot be explained by the shunting effect alone, even if the sandwich model postulated by Bamberg et al. (Biophys. Struct. Mech., 5 (1979) 277) is invoked. We suspect that the incorporation of bacteriorhodopsin into the artificial black lipid membrane may be more complete than initially believed.

Circulatory Thyroglobulin Threshold in Suppressor Activation

Murine experimental autoimmune thyroiditis (EAT) has served as a model for Hashimoto’s thyroiditis, and mouse thyroglobulin (MTg) as a model for self antigen of the thyroid in genetically susceptible (MHC-associated) individuals1-. EAT is induced readily when autoreactive inducer/helper T cells (TI) are stimulated by MTg given repeatedly2 or with an adjuvant, such as lipopolysaccharide (LPS)3. However, the preinjection of 100-200 yg MTg or thyroid-regulating hormones (TSH or TRH) activates suppressor mechanisms which interfere with EAT induction4. Both regimens elevate the levels of circulating MTg for a short interval.