Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.     

Coexpression of Aspartic Proteinases and Human Leukocyte Antigen-DR in Human Transplanted Lung

Aspartic proteinases have recently been shown to be implicated in antigen processing. We explored the expression of two aspartic proteinases, cathepsins E and D, and of human leukocyte antigen-DR (HLA-DR) molecules in a consecutive series of 80 transbronchial biopsies from transplanted lungs. For controls, we studied five normal donor lungs (not suitable for transplantation on account of thoracic trauma) and macroscopically normal areas of three cancer-affected lungs. Two of the five unsuitable donor lungs showed minimal inflammatory changes. Macroscopically normal samples from the three cancerous lungs showed mild and focal inflammatory infiltrates. In histologically normal lungs, HLA-DR expression was limited to professional antigenpresenting cells. Macroscopically normal lung samples with minimal inflammatory changes from both donor and cancer lungs showed variable HLA-DR expression by alveolar and bronchial epithelial cells and by endothelial cells. All transplanted lung biopsies showed HLA-DR expression by epithelial (alveolar and bronchial) and endothelial cells, with a trend for increased positivity in acute rejection. Cathepsin E was restricted to Clara and to rare bronchus-associated lymphoid tissue-related epithelial cells in histologically normal lung samples, whereas minimal de novo cathepsin E expression by rare alveolar pneumocytes was noted in control lung samples exhibiting minimal inflammatory changes. In all transplanted lung biopsies, cathepsin E was diffusely expressed de novo by hyperplastic alveolar epithelial cells, regardless of the presence or degree of rejection. Cathepsin D was expressed only by alveolar macrophages and by ciliated bronchial cells of normal, minimally inflamed, and transplanted lungs. In transplanted lung, Clara cells and several hyperplastic alveolar pneumocytes coexpressed HLA-DR and cathepsin E, whereas all alveolar macrophages and a few ciliated cells coexpressed cathepsin D and HLA-DR The present investigation suggests that the de novo expression of cathepsin E and HLA-DR by hyperplastic alveolar pneumocytes of transplanted lung may be crucial for antigen processing and presentation to recipient competent T cells, and thus for the triggering of the immune-inflammatory cascade that leads to rejection.     

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH₂-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Role of BCR affinity in T cell dependent antibody responses in vivo

Antibody affinity for antigen is believed to govern B lymphocyte selection during T-dependent immune responses. To examine antibody affinity in T cell dependent immune responses, we compared mice that carry targeted V(H)B1-8 antibody genes with high or low antigen-binding affinity. We found that high- and low-affinity B cells had the same intrinsic capacity to respond to antigen, but in experiments where limiting numbers of high- and low-affinity B cells were mixed in wild-type recipient mice, only the high-affinity B cells accumulated in germinal centers (GCs). In GCs, high-affinity B cells accumulated fewer V(H) somatic mutations than low affinity B cells. This effect was due to selections as the frequency of mutation in noncoding immunoglobulin gene DNA is the same in high- and low- affinity B cells. Thus, B cells recruited to the GC appeared to undergo a fixed mutation program, regardless of initial B cell receptor affinity. We conclude that in addition to the selection that occurs in GCs, stringent selection for high-affinity clones is also imposed in the early stages of the T cell dependent immune response in vivo.     

Interleukin-10 secreted by B-1 cells modulates the phagocytic activity of murine macrophages in vitro

As demonstrated previously in our laboratory, B-1 cells migrate from the peritoneal cavity of mice and home to a distant site of inflammation to become macrophage-like cells. However, the influence that these cells might have on the kinetics and fate of the inflammatory process is not known. Considering that macrophages are pivotal in the inflammatory reaction, we decided to investigate the possible influence B-1 cells could have on macrophage activities in vitro. Our results show that peritoneal macrophages from Xid mice, a mouse strain deprived of B-1 cells, have higher phagocytic indexes for zymozan particles when compared with macrophages from wild-type mice. Moreover, macrophages from wild-type mice have a lower ability to release nitric oxide and hydrogen peroxide when compared with macrophages from Xid mice. Experiments using cocultures of B-1 cells and macrophages from Xid mice in transwell plates demonstrated that B-1 cells down-regulate macrophage activities. These observations also indicate that this phenomenon is not due to a physical interaction between these two cell populations. As B-1 cells are one of the main sources of interleukin (IL)-10, we demonstrate in this study that adherent peritoneal cells from Xid mice produce significantly less amounts of this cytokine in culture when compared with IL-10 production by cells from wild-type mice. When B-1 cells from IL-10 knock-out mice and macrophages from wild-type mice were cocultured in transwell plates, the phagocytic index of macrophages was not altered demonstrating that B-1 cells can influence the effector functions of macrophages in vitro via IL-10 secretion.     

Lymphopenia in acute nitrofurantoin pleuropulmonary reactions

Each of 5 patients with acute nitrofurantoin pleuropulmonary reactions had profound lymphopenia and 4 had eosinophilia developing early in the clinical course after the drug was withdrawn. The 2 patients tested had only one third of the normal numbers of E rosettes (T lymphocytes) in the peripheral blood during recovery. Lymphoblastic transformation tests with purified nitrofurantoin were done in 3 patients and all of them were negative; responses to phytohemagglutinin, concanavalin A, and pokeweed were decreased but still normal. The diagnosis of various nitrofurantoin hypersensitivity reactions relies on clinical data. The mechanisms of these reactions presently remain unclear.     

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.