Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

Abstract

C57Bl/6 female mice were infected with an intrapulmonary dose of 2.5 x 10(4) BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1, 2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4-6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3+ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1+) per lung increased by about 50%, 5-6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNgamma producing non-T cells outnumbered T cells producing IFNgamma. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNgamma producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNgamma response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNgamma, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine.

Inactivation of CD4+CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load

“…To determine whether Treg are present in the lung and whether they accumulate during mycobacterial lung infection, we measured the number of CD4 + CD25 + Foxp3 + Treg in the lung after an intranasal challenge with BCG. BCG is attenuated M. bovis that causes a pulmonary infection in mice similar to that of virulent mycobacteria as it results in the infiltration of lymphocytes, macrophages and granulocytes to the lung, 21 and IFN‐γ production is required for control of the infection 22 . We found that over the first 14 days of BCG infection the total number of Treg in the lung almost trebled in control mice treated with rat IgG (Figure 1A), indicating that this cell subset is indeed present in the normal lung (day 0) and increases significantly during a mycobacterial lung infection.…”

Innate Resistance to Tuberculosis in Man, Cattle and Laboratory Animal Models: Nipping Disease in the Bud?

“…The role of NK cells in the innate response to Mtb is evidenced by their increased number and activated status in the lungs of infected mice (Saxena et al, 2002;Feng et al, 2006) and their innate effector function is illustrated by their capacity to lyse Mtbinfected human monocytes (Vankayalapati et al, 2005). This capacity is due to their intracellular preformed perforin and granulysin granules, which can also directly kill extracellular mycobacteria and restrict mycobacterial growth in an apoptosis-dependent but Fas/Fas ligand (FasL) independent manner (Stenger et al, 1998;Brill et al, 2001;Millman et al, 2008).…”

Rituximab-CD20 Complexes Are Shaved from Z138 Mantle Cell Lymphoma Cells in Intravenous and Subcutaneous SCID Mouse Models

“…Cell suspensions were prepared from lungs or solid tumors by digestion with a mixture of collagenase, hyaluronidase, elastase, and DNase (37,38), purified by density gradient centrifugation (39), washed, and reconstituted in 2 mg/ml mouse IgG in BSA-PBS. Samples were stained as described below, and analyzed by flow cytometry, following procedures used previously to examine B cells from CLL patients (21,25).…”