John W. Moorhead

Suppressive mechanisms involving sensitization and tolerance in contact allergy.

Cotitact sensitivity has been studied extensively in man and experitnental animals. It represents an example of antigen-specific delayed hypersensitivity and was thought to provide a simple model of this type of reaction. Within recent years, there has been an acceleration of research in this area. Increasing data have accumulated to show that contact sensitivity is in reality a very complex phenomenon. Furthermore, current interest in the regulation of immunologic processes has prompted detailed studies of the ways in which contact sensitivity is controlled. This paper will summarize the work which has been carried out in our laboratory since 1974 on contact sensitivity to DNFB in mice. The material will be centered around the suppressive mechanisms which have been uncovered in both tolerance to contact sensitivity and also in the appearance, magnitude and duration of contact allergy itself. Although other control mechanisms (e.g. tolerance without suppression) may also exist, they will not be discussed at length here. The work will primarily analyze our own experiments, but this should not be interpreted to ignore or slight the very important contributions made previously by others (Chase 1967, DeWeck & Frey 1966, Eisen et al. 1952, Polak & Turk 1974, Asherson & Zembala 1976).

Allogeneic grafts of fetal dopamine neurons: immunological reactions following active and adoptive immunizations

To define the importance of adoptive sensitization and duration of graft residence on transplant alloimmunization, behavioral and histochemical parameters were examined in unilaterally 6-OHDA-lesioned F344 rat hosts which received fetal ventral mesencephalic (VM) grafts from Wistar-Furth (WF) donors. In all animals which showed increased rotations after alloimmunization, increased numbers of T cell receptor (TcR) positive, CD8+ lymphocytes were detected in the grafts. In addition, an increased density of class I MHC antigens was seen in the graft and in the adjacent host brain. Lesser numbers of CD4+, CD11b+, and MHCII+ positive elements were also seen. Perivascular cuffing was often found in actively immunized animals. An increase in TcR+ and MHC class I+ elements was also seen in animals only adoptively immunized. The tyrosine hydroxylase positive graft area was also markedly reduced in actively immunized animals and the extent of reduction correlated with the number of cells used for immunization. These studies indicate that established allografts can evade rejection as long as host lymphocytes are not activated against graft alloantigens. In addition, increasing graft residence time in the host and adoptive immunization render the graft more susceptible to subsequent rejection.

Allogeneic grafts of fetal dopamine neurons: behavioral indices of immunological interactions.

Fetal central nervous system transplants to the adult brain have been utilized to understand brain connectivity and as replacement therapy in Parkinsons disease (PD). Here we use fetal brain allografting in the rat unilaterally depleted of dopamine, a unilateral model of PD, and apomorphine-induced rotations as an index of graft functional status while peripherally manipulating the hosts alloimmune status. This system allows the investigator to examine, dynamically, host-allograft interactions in the brain under differing states of alloimmunoreactivity without the need to biopsy or sacrifice the animal. In addition to this novel application, we established that brain allografts are differentially susceptible to immunologic attack depending upon the grafts duration of residence in the host brain. Increasing residence time increases graft rejectability to peripheral allosensitization. Passive immunization also sensitizes the host to subsequent graft rejection. Lastly, simple host alloimmunocompetence is necessary but not sufficient to cause fetal graft rejection, defined as a return of apomorphine-induced rotations.

Microglial Cell Responses to Fetal Ventral Mesencephalic Tissue Grafting and to Active and Adoptive Immunizations

Microglia express cytokines, major histocompatibility (MHC) loci, and several other immunologically important constituents. The aim of this study was to detect immunological responses of microglial cells following allogeneic dopaminergic transplantation using active and adoptive immunizations. Adult inbred Fisher 344 (F344 RT1) rats were unilaterally dopamine (DA) depleted in striatum by injection of 6-hydroxydopamine. The degree of degeneration was assessed by recording the rotational response to apomorphine. Fetal ventral mesencephalic tissue containing DA neuroblasts from Wistar-Furth (WF, RT1u) rat donors (9-12 mm CRL) were later implanted in striatum on the lesioned side. Lymph nodes and spleen cells were collected aseptically, resuspended, and diluted for isovolumetric injections. Animals selected for active immunization were injected intraperitoneally with varying amounts of WF lymphocytes. Animals selected for adoptive immunization (transferred immunity) were intraperitoneally injected with 10(8) F344 lymphocytes prepared from animals actively immunized 3 weeks previously. Monoclonal antibodies against CD4 (OX38), CD8 (OX8), CD11b (OX42), MHC class I (OX18), monomorphic MHC class II (OX-6), and ED1 and polyclonal antibodies against tyrosine hydroxylase (TH) were used for immunohistochemistry. We found that the degree of ED1-positive cell proliferation was well correlated to the immunization patterns. Groups that were actively immunized with or without prior adoptive immunization had a larger amount of reactive microglial proliferation. ED1 immunohistochemistry revealed patterns of immunolabeling of engrafted areas: 8-12 weeks after grafting in nonimmunized and adoptively immunized groups reactive microglial proliferation occurred only at the graft periphery. Active and adoptive + active immunization led to ED1-IR within the grafts themselves. At early stages nonimmunized groups had an ED1 pattern which was partially inside the grafts. At early time points nonimmunized groups contained ameboid microglial cells within the grafts which disappeared at later stages and were absent in the immunized groups. ED1-positive ameboid microglial cells within the grafts may be of graft origin and constitute a part of a continued normal development of the fetal tissue.

Intranigral transplantation of solid tissue ventral mesencephalon or striatal grafts induces behavioral recovery in 6-OHDA-lesioned rats

Parkinsons disease (PD) is characterized by a degeneration of the dopamine (DA) pathway from the substantia nigra (SN) to the basal forebrain. Prior studies in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats have primarily concentrated on the implantation of fetal ventral mesencephalon (VM) into the striatum in attempts to restore DA function in the target. We implanted solid blocks of fetal VM or fetal striatal tissue into the SN to investigate whether intra-nigral grafts would restore motor function in unilaterally 6-OHDA-lesioned rats. Intra-nigral fetal striatal and VM grafts elicited a significant and long-lasting reduction in apomorphine-induced rotational behavior. Lesioned animals with ectopic grafts or sham surgery as well as animals that received intra-nigral grafts of fetal cerebellar cortex showed no recovery of motor symmetry. Subsequent immunohistochemical studies demonstrated that VM grafts, but not cerebellar grafted tissue expressed tyrosine hydroxylase (TH)-positive cell bodies and were associated with the innervation by TH-positive fibers into the lesioned SN as well as adjacent brain areas. Striatal grafts were also associated with the expression of TH-positive cell bodies and fibers extending into the lesioned SN and an induction of TH-immunolabeling in endogenous SN cell bodies. This finding suggests that trophic influences of transplanted fetal striatal tissue can stimulate the re-expression of dopaminergic phenotype in SN neurons following a 6-OHDA lesion. Our data support the hypothesis that a dopaminergic re-innervation of the SN and surrounding tissue by a single solid tissue graft is sufficient to improve motor asymmetry in unilateral 6-OHDA-lesioned rats.

Strong foreign promoters contribute to innate inflammatory responses induced by adenovirus transducing vectors

E1-deleted adenovirus (FG Ad) transducing vectors are limited for use in vivo by their induction of strong innate and adaptive inflammatory responses. We have examined the contribution of the transgene cassette, particularly the foreign promoter driving transgene expression, in the induction of innate inflammation using a mouse ear model in which swelling is measured as a sensitive surrogate marker of the total innate inflammatory response. The commonly used cytomegalovirus major immediate early (CMV) promoter led to high-level swelling that was independent of transgene expression, while the Rous sarcoma virus and human ubiquitin C promoters led to intermediate levels of swelling and the Ad E1A promoter or no promoter led to equally low levels of swelling. Significant swelling was induced by a virus in which the E1A promoter directed pIX expression, supporting the possibility that activation of expression of Ad genes retained in the vector plays an important role in the inflammatory response. Taken together, our findings support the idea that strong foreign promoters likely play the limiting role in the induction of innate and adaptive immune responses that limit the duration of transgene expression after transduction by FG Ad vectors.

Anti-immunoglobulin stimulation of murine lymphocytes: VI. Differential responses of complement receptor positive (CR+) and CR− B-cell subsets to anti-IgM antibodies☆

Abstract Previous studies have shown that splenic B cells from aged mice (>7 months) proliferate in vitro when stimulated with intact rabbit anti-IgM or Fab′ 2 anti-IgM antibodies. The response to intact anti-IgM is macrophage dependent whereas the response to Fab′ 2 anti-IgM is macrophage independent. In the present studies, experiments were done to determine if the differential macrophage requirement is due to subsets of B cells responding to the different molecular forms of the anti-IgM antibodies. Spleen cells from aged mice were separated into populations enriched for complement receptor negative (CR − ) and CR + B cells by EAC rosetting and gradient centrifugation. The enriched populations were then tested for their proliferative response to intact and Fab′ 2 anti-IgM. Compared to unseparated spleen cells, CR − B cells show enhanced responses to intact anti-IgM and reduced responses to Fab′ 2 anti-IgM. In contrast, CR + B cells do not respond to intact anti-IgM molecules; but their response to Fab′ 2 anti-IgM is significantly enhanced compared to unseparated cells. Unresponsiveness appears to be mediated via interactions of Fc receptors on the CR + B cells with the Fc portion of the anti-IgM molecule. Addition of small amounts of intact anti-IgM completely inhibits the Fab′ 2 -induced proliferative response. Collectively, these results indicate that CR − and CR + B cells have different signaling requirements for activation and that the subsets differ with respect to their sensitivity to Fc receptor-mediated inhibition.

Negative feedback regulation of contact sensitivity to DNFB by autoanti-idiotypic antibody.

Contact sensitivity to 2,4-dinitrofluorobenzene is maximal six days after sensitization but declines rapidly, due to autoanti-idiotypic antibodies produced by the host. The studies presented here indicate that this down regulation by anti-Id is a C-independent active process involving a subset of Ia+ T cells in the immune lymph node cell population. Depleting immune LN cells of Ia+ T cells renders them insensitive to inhibition by anti-Id alone, although the same population is inhibited by treatment anti-Id plus C. This cell population is rendered sensitive to inhibition by anit-Id alone by adding untreated DNFB-sensitized LN cells but not by adding normal LN cells. Further studies showed that suppression by anti-Id-activated Ia+ T cells occurs locally at the skin test site and is antigen nonspecific. These data indicate that the natural regulation of CS to DNFB by autoanti-Id antibodies involves a negative feedback regulatory loop.