Cell-secreted Gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8+ cytotoxic T lymphocytes in the intestinal mucosa.

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyer's patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.

The presence of functional mannose receptor on macrophages at the maternal-fetal interface.

BACKGROUND: The mannose receptor (MR) is involved in the initiation of the immune response and regulation of homeostasis during inflammation and tissue remodeling. METHODS: Distribution, endocytosis and possible natural ligand tumor associated glycoprotein-72 (TAG-72) for the MR have been examined by immunohistology, immunocytochemistry and flow cytometry at the maternal-fetal interface, characterized by extensive tissue remodeling. RESULTS: Contrary to disseminated distribution of the MR positive (MR+) cells in term placenta, the MR+ cells of early pregnancy decidua intimately surrounded glands and followed tissue distribution of CD14 positive cells. The mannose receptor was present on freshly isolated first trimester decidual mononuclear cells and distributed mostly on macrophages (77.08 +/- 10.55%, mean +/- SD). The expression of the MR on CD14 positive cells decreased following 18 h culture (P < 0.01) and was accompanied by the reduction of fluorescein isothiocyanate (FITC)-dextran uptake. PAM-1 anti-MR antibody, mannan and TAG-72 reduced FITC-dextran uptake by decidual macrophages. CONCLUSIONS: These data indicate that the MR+ macrophages, surrounding early decidual glands, are able to internalize ligands for carbohydrate recognition domain of the receptor, including decidual secretory phase mucin TAG-72.

Perforin expression in peripheral blood lymphocytes and skin-infiltrating cells in patients with lichen planus.

BACKGROUND: Current evidence suggests that lichen planus is a T-cell-mediated autoimmune disease in which cytotoxic mechanisms have been poorly investigated. OBJECTIVES: We investigated the expression of perforin in subpopulations of peripheral blood lymphocytes (PBL) in exacerbation and remission phases of the disease as well as in skin lesions. METHODS: We performed a simultaneous detection of perforin (intracellular molecule) and cell surface antigens on PBL by flow cytometry, and skin lesions were investigated by immunohistochemistry. RESULTS: The most interesting finding was a significant increase of perforin expression in cytotoxic T lymphocytes (CD3+ perforin+ cells) in the exacerbation phase of disease (P < 0.05), which was mostly located in the CD8+ subpopulation (CD8+ perforin+) (P < 0.01). Using immunohistochemistry we confirmed the infiltration of T lymphocytes in skin lesions, especially of CD4+ and CD8+ phenotypes, compared with uninvolved (P < 0.05) and healthy skin (P < 0.01). The expression of perforin was also significantly higher in lesional skin compared with nonlesional and healthy skin (P < 0.05). CONCLUSIONS: Our results clearly show the upregulation of perforin expression in peripheral blood as well as in lesions of patients with lichen planus and therefore suggest an important role for perforin in this autoimmune disease.

Assessment of perforin expression in peripheral blood lymphocytes in psoriatic patients during exacerbation of disease.

There are very few data concerning the role played by cell-mediated cytotoxicity, particularly at the molecular level, in the course of psoriasis. Both cytotoxic T lymphocytes (CTL) and natural killer cells contain in their granules the cytolytic protein perforin, a mediator in cell-mediated cytotoxicity reactions. The aim of this study was to analyze perforin expression in various sets and subsets of perforin-positive peripheral blood lymphocytes in 17 patients with chronic psoriasis vulgaris in the exacerbation phase. The results were compared with those of an age- and sex-matched healthy control group (n = 21). Perforin (intracellular antigen) and cell surface antigens were detected using the simultaneous double-staining method. We found a significant increase in perforin (P) expression in the patient group for CTL (CD3+P+ cells), which are located mostly in the CD8+ population of T lymphocytes (CD8+P+).

Progesterone directly and indirectly affects perforin expression in cytolytic cells.

PROBLEM: Decidual lymphocytes (DL) expressing the cytolytic molecule perforin represent approximately 55% of DL in the first trimester of human pregnancy. Progesterone dominates this phase of pregnancy and controls the production of uterine cytokines and growth factors. The aim of this study was to investigate the role of progesterone and progesterone-induced blocking factor (PIBF) on perforin expression in DL and peripheral blood lymphocytes (PBL). METHOD OF STUDY: Perforin expression was analyzed in PBL and DL incubated either in culture medium or with decidual adherent cells (DAC) and peripheral blood adherent cells (PBAC) and their supernatants with or without progesterone or PIBF. Perforin was detected by flow cytometry in PB and in decidual first trimester pregnancy lymphocytes. RESULTS: Progesterone in high concentrations directly affects perforin expression in DL but not in PBL. Progesterone in a concentration dependent manner indirectly blocks perforin expression in DL and PBL cultured with adherent cells or their supernatants. PIBF blocked upregulation of perforin expression of DL cultured with DAC, but none of those cultured with PBAC. Similarly, PIBF was inefficient when PBL or DL were cultured with PBAC. CONCLUSION: Progesterone present in a high concentration locally at the maternal-fetal interface modulates perforin expression in the first trimester pregnancy DL.

Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells.

PROBLEM: We have shown that addition of decidual adherent cells (DAC) to the culture of decidual lymphocytes (DL) prevents the downregulation of perforin expression in these cells. Because DAC are a mixture of various cell populations, the aim is to analyze immunophenotypic characteristics of DAC and to determine which cell population is involved in the regulation of perforin expression. METHOD OF STUDY: First trimester pregnancy decidual cells were obtained by enzymatic tissue digestion. Decidual cells and peripheral blood lymphocytes (PBL) were centrifuged on Ficoll-Hypaque density gradient and cultured overnight to obtain adherent cells, which were analyzed by flow cytometry and immunocytochemically. RESULTS: Almost all peripheral blood adherent cells (PBAC) (ca 90%) expressed monocyte/macrophage markers but only 10-20% of DAC. The rest of DAC expressed markers of stromal cells. HLA-DR depleted population of DAC (stromal cells only) could not prevent downregulation of perforin expression in cultured DL and PBL. CONCLUSION: Decidual macrophages are involved in the regulation of perforin expression in DL.

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.

Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells.

In this study a flow cytometric technique for detecting cytoplasmic perforin (P) has been used to quantify age-related changes in perforin expression in human peripheral blood lymphocytes (PBL). Proportions of P+ lymphocytes increased after birth, but declined rapidly after the age of 70 years. This was true for both T cells and CD16(+) and CD56(+) natural killer (NK) cells. Children showed in addition to high levels of perforin positive CD8(+) cells a much higher proportion of CD4(+)P+ cells than the other age groups. In elderly individuals there was also a highly significant reduction in mean levels of perforin per cell as compared with all other groups (P < .05 to .001). Adult women had consistently higher mean levels of perforin per cell than adult men for all P+ cell phenotypes. Functional tests clearly showed the deficiency in early spontaneous cytotoxic potential of PBL from elderly persons due to relative P deficiency, which can be corrected by stimulation of cytolytic cells with target cells and interleukin-2 (IL-2). The deficiency in cytolytic activity on the contact with target cells may have implications for antiviral and antitumor immunity in elderly persons.