David A. Schmitt

Human Colonic Myofibroblasts Promote Expansion of CD4+ CD25high Foxp3+ Regulatory T Cells

BACKGROUND & AIMS Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells. METHODS Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohns disease, were used to assess activation of the Treg cells. RESULTS Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-β and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3. CONCLUSIONS CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases.

Role of Gastric Epithelial Cell-Derived Transforming Growth Factor β in Reduced CD4+ T Cell Proliferation and Development of Regulatory T Cells during Helicobacter pylori Infection

ABSTRACT Gastric epithelial cells (GECs) express the class II major histocompatibility complex (MHC) and costimulatory molecules, enabling them to act as antigen-presenting cells (APCs) and affect local T cell responses. During Helicobacter pylori infection, GECs respond by releasing proinflammatory cytokines and by increasing the surface expression of immunologically relevant receptors, including class II MHC. The CD4+ T cell response during H. pylori infection is skewed toward a Th1 response, but these cells remain hyporesponsive. Activated T cells show decreased proliferation during H. pylori infection, and CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) are present at the site of infection. In this study, we examined the mechanisms surrounding the CD4+ T cell responses during H. pylori infection and found that transforming growth factor β (TGF-β) plays a major role in these responses. GECs produced TGF-β1 and TGF-β2 in response to infection. Activated CD4+ T cells in culture with H. pylori-treated GECs were decreased in proliferation but increased upon neutralization of TGF-β. Naïve CD4+ T cell development into Tregs was also enhanced in the presence of GEC-derived TGF-β. Herein, we demonstrate a role for GEC-produced TGF-β in the inhibition of CD4+ T cell responses seen during H. pylori infection.

Inhibition of burn-associated suppressor cell generation by glycyrrhizin through the induction of contrasuppressor T cells

The inhibitory effect of glycyrrhizin (GR), an anti‐inflammatory Chinese herbal drug extracted from licorice roots, on the generation of suppressor T cells in thermally injured mice (TI‐mice) was investigated. The burn‐associated suppressor T cell (BTs cell) activity was demonstrated in splenic mononuclear cells (SMNC) from mice 2 to 8 days after thermal injury when suppressor cell activity was assayed in a one‐way mixed lymphocyte reaction. However, when TI‐mice were treated with GR, SMNC harvested 6 days after thermal injury showed no suppressor cell activity. This activity of GR demonstrated a dose‐response effect, with a dose of 10 mg/kg exhibiting peak levels of the activity. Since GR had no direct inactivating activities against BTs cells in vitro, the inhibitory effect of SMNC, derived from TI‐mice treated with GR, on the activity of BTs cells was examined in the same mixed lymphocyte reaction system, and the results showed that the SMNC from GR‐treated mice 6 days after thermal injury counteracted the activity of BTs cells. The type of cell responsible for this inhibition of BTs cell activities was a CD3+, L3T4+, Vicia villosa lectin‐adherent T cell with the same phenotypic properties previously exhibited by contrasuppressor cells. These results suggest that GR may regulate the generation of BTs cells through the induction of contrasuppressor cells. Since there are many reports describing septic infections due to the appearance of BTs cells in postburn patients, it may be possible to apply GR or blood preparations containing contrasuppressor cell populations induced by GR in healthy volunteers into immunosuppressed burn patients to avoid infections.

Antiviral effects of recombinant human tumor necrosis factor-alpha in combination with natural interferon-beta in mice infected with herpes simplex virus type 1

The protective effects of combination therapy utilizing recombinant human TNF-alpha (rTNF-alpha) and natural murine interferon-beta (IFN-beta) in mice infected with herpes simplex virus type 1 (HSV-1) was investigated. Mice treated with rTNF-alpha alone at all of the doses tested (a single i.v. administration, 2.3-2,300 micrograms/kg; multiple i.p. administrations 0.4-250 micrograms/kg) as well as mice that received IFN-beta alone at doses of 16 x 10(4) U/kg or less resulted in a 0% survival rate. Combination therapy consisting of a single administration of rTNF-alpha (230 and 23 micrograms/kg) and multiple administrations of IFN-beta (4 x 10(4) U/kg) resulted in a 40% and 60% survival rate. Multiple treatments of infected mice with rTNF-alpha (50 and 10 micrograms/kg) in combination with IFN-beta (4 x 10(4) U/kg) resulted in 50% and 70% survival rates, respectively. These results suggest that the combination therapy of rTNF and natural murine IFN-beta produce synergistic protective effects in mice infected with a lethal amount of HSV-1.

Preventive Effect of A Synthetic Immunomodulator, 2-Carboxyethylgermantum Sesquioxide, on the Generation of Suppressor Macrophages in Mice Immunized with Allogeneic Lymphocytes

The effect of 2-carboxyethylgermanium sesquioxide (Ge-132) on the generation of splenic suppressor macrophages (S-M phi) in C3H/He mice (H-2k) immunized with allogeneic spleen cells from C57Bl/6 mice (H-2b) was investigated. We have previously demonstrated that S-M phi expressing I-J antigen, which appeared during alloimmunization, inhibited cytotoxic T lymphocyte (CTL) generation in the MLR and the elimination of these S-M phi before subjection to the MLR resulted in more effective generation of CTL. The CTL activity, which was determined in vivo by the Winns test, was markedly enhanced when immunized mice received a 100 mg/kg dose of Ge-132. The compound was found to be the most efficacious when injected simultaneously with the immunization. The activity of allospecific CTL co-cultured with M phi fractions obtained from immunized mice in a 4-h 51Cr-release assay was shown to be 31% lysis of the target cells as compared with 90% lysis of the target cells in effector cells co-cultured with normal M phi fractions. In contrast, effector cells co-cultured with M phi fractions from Ge-132-treated immunized mice lysed 95% of the target cells. Analysis of the level of I-J antigen expression on macrophages (M phi) obtained from mice 7 days after immunization revealed a > 2.5-fold increase, whereas I-A antigen expression remained constant when compared with splenic M phi from naive mice. In contrast, the opposite effect on I-J and I-A antigen expression was observed in splenic M phi from alloimmunized mice treated with Ge-132. These results suggest that Ge-132 could regulate CTL generation in alloimmunized mice by preventing the generation of I-J+ S-M phi.

Immunomodulating activities of orally administered SMANCS, a polymer-conjugated derivative of the proteinaceous antibiotic neocarzinostatin, in an oily formulation

Immunomodulatory effects of oily formulated SMANCS, a polymer conjugated derivative of the proteinaceous antibiotic neocarzinostatin, after oral administrations to mice was investigated. The oral administrations of SMANCS, dissolved in medium-chain triglyceride containing phosphatidylcholine and polyglycerine dioleate (oily SMANCS), resulted in: (1) augmentation of NK cell activity in naive mice, (2) activation of macrophage cytostasis in naive mice, (3) enhancement of the generation of cytotoxic T-lymphocytes in mice immunized with allogeneic lymphocytes, (4) production of circulating interferon in naive mice, and (5) increase of delayed-type hypersensitivity response in mice immunized with sheep red blood cells. The degree of immunomodulation orally stimulated with oily SMANCS was similar to that of the immunomodulation induced by i.v. or i.p. administrations of aqueous formulated SMANCS (aqueous SMANCS). Although SMANCS is a protein drug that may be digestable by various enzymes present in the stomach, in the present study immunomodulating activities of SMANCS were clearly demonstrated when an oily formulation of the compound was administered to mice orally. Since aqueous SMANCS administered parenterally and oily SMANCS administered orally exhibit the same immunomodulatory activities and the former has demonstrated antitumor activity in man and animals, the latter may possess this same antitumor activity.