Ronald J. Pettis

Aerosol delivery of muramyl dipeptide to rodent lungs

Tuberculosis is the single most serious infectious disease worldwide. The respiratory tract is the primary site of infection by Mycobacterium tuberculosis (MTB). A number of immunogenic components of the cell wall of MTB, if delivered to the lungs as aerosols, can be used to study the local immune response. The site of deposition of these aerosols can be employed to control their residence time in the lungs. Muramyl dipeptide (MDP) aerosols were delivered to alveolar macrophages in the lungs of rodents. Guinea pig macrophages harvested by bronchoalveolar lavage were examined by differential interference contrast microscopy for morphological changes indicative of activation. Bronchoalveolar lavage fluid was analyzed for the presence of alkaline phosphatase, lactate dehydrogenase, N-acetyl-glucosaminidase (NAG), and total protein content. Rat alveolar macrophages were studied for the production of nitric oxide, by induction of nitric oxide synthase. Twenty-four hours following exposure to an aerosol of MDP, alveolar macrophages exhibited morphological characterstics (spreading and pseudopodia), enzyme activity (NAG 50% above control), and production of the reactive intermediate nitric oxide. Rat macrophages subjected to aerosol exposure to MDP when challenged with a second dose of MDP or lipopolysaccharide exhibited a linear dose response as measured by nitric oxide production. These studies indicate that the topical delivery of an MTB bacterial cell wall component. muramyl dipeptide, results in activation of alveolar macrophages. This approach may be useful in elucidating elements of the immune response to MTB.

TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4–25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFβ in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes.Key messagessHA promotes lymphangiogenesis primarily through increased LEC proliferationsHA induces proliferation in a narrow concentration window due to upregulated TGFβSmaller HA oligosaccharides more potently induce proliferation than larger onesVEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHASialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC