Steroid-Resistant Lymphatic Remodeling in Chronically Inflamed Mouse Airways

Angiogenesis and lymphangiogenesis participate in many inflammatory diseases, and their reversal is thought to be beneficial. However, the extent of reversibility of vessel remodeling is poorly understood. We exploited the potent anti-inflammatory effects of the corticosteroid dexamethasone to test the preventability and reversibility of vessel remodeling in Mycoplasma pulmonis-infected mice using immunohistochemistry and quantitative RT-PCR. In this model robust immune responses drive rapid and sustained changes in blood vessels and lymphatics. In infected mice not treated with dexamethasone, capillaries enlarged into venules expressing leukocyte adhesion molecules, sprouting angiogenesis and lymphangiogenesis occurred, and the inflammatory cytokines tumor necrosis factor and interleukin-1 increased. Concurrent dexamethasone treatment largely prevented the remodeling of blood vessels and lymphatics. Dexamethasone also significantly reduced cytokine expression, bacterial burden, and leukocyte influx into airways and lungs over 4 weeks of infection. In contrast, when infection was allowed to proceed untreated for 2 weeks and then was treated with dexamethasone for 4 weeks, most blood vessel changes reversed but lymphangiogenesis did not, suggesting that different survival mechanisms apply. Furthermore, dexamethasone significantly reduced the bacterial burden and influx of lymphocytes but not of neutrophils or macrophages or cytokine expression. These findings show that lymphatic remodeling is more resistant than blood vessel remodeling to corticosteroid-induced reversal. We suggest that lymphatic remodeling that persists after the initial inflammatory response has resolved may influence subsequent inflammatory episodes in clinical situations.Chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, Crohn''s disease, and skin lesions in psoriasis are accompanied by a spectrum of remodeling changes in the microvasculature.^1–5 In inflamed tissues, blood vessels undergo angiogenesis and remodeling to change their structure and function. Existing capillaries become leakier and abnormally enlarged in diameter and show venular features.^6–8 The capillary-to-venule transformation increases the amount of vasculature capable of supporting leukocyte adhesion and migration in response to inflammation stimuli. Conventional sprouting angiogenesis also occurs, usually later than the capillary enlargement. Lymphatic vessels also proliferate from existing lymphatic endothelial cells by sprouting lymphangiogenesis and undergo remodeling to compensate for the extra need for drainage in the inflamed tissues and trafficking of leukocytes, thereby contributing to the development of pathophysiology.^9–11Whereas the remodeling and growth of vessels in inflammation has been documented in an increasing number of studies, the reversibility of vessel changes is not well understood. Relatively little is known about whether the newly grown lymphatics can regress after they have formed at sites of inflammation, and, if so, how quickly. Infection of the airways by the natural rodent respiratory tract pathogen Mycoplasma pulmonis results in persistent vessel changes and life-long airway inflammation.^12,13 Similar airway vessel changes and chronic inflammation are also common symptomatic features found in human asthma and chronic bronchitis.¹¹ In M. pulmonis infection, the robust growth and remodeling of blood vessels and lymphatics are driven by a cascade of immune responses to sustained bacterial infection.¹⁴ Gene profiling experiments have shown that many inflammatory molecules are up-regulated in M. pulmonis-infected lungs and that many interrelated pathways are likely to drive downstream endothelial cell remodeling.^15–17 In this model, partial reversal of enlarged blood vessel diameter occurs after corticosteroid treatment for 1 week.⁷ Elimination of infecting bacteria with antibiotics for 4 weeks fully reverses the enlargement of blood vessels but results in only a partial reversal of the newly formed lymphatic network.¹⁰The aim of this study was to further clarify the prevention and reversibility of all aspects of blood vessels and lymphatics associated with chronic airway inflammation after M. pulmonis infection. To achieve this purpose, we used the corticosteroid dexamethasone as a powerful tool to repress a wide array of inflammatory mediators, including chemokines, cytokines, growth factors, receptors, and adhesion molecules.^18–20 In addition to its broad-spectrum anti-inflammatory function, dexamethasone can down-regulate the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C.^21,22 Dexamethasone can also reduce angiopoietin-2 expression in cultured endothelial cells.²³ We reasoned that a study with a potent anti-inflammatory and anti-angiogenic agent would help in interpreting the maximum degree of prevention and reversibility and would be a useful basis for future studies with more selective agents.We performed two treatment studies with dexamethasone, beginning either concurrently at the time of inoculation or after every aspect of vessel changes had already been established. In each study, we examined the time course and extent of vessel changes. We also examined the effects of dexamethasone treatment on the M. pulmonis-driven immune responses. We found that dexamethasone treatment prevented the vessel changes and the associated inflammatory responses induced by M. pulmonis infection more effectively than it reversed them. Delayed treatment reversed remodeled blood vessels almost to pathogen-free conditions and regressed angiogenic and lymphangiogenic sprouting. In contrast, newly formed lymphatics persisted and were remarkably resilient to regression. Furthermore, associated inflammatory responses were reduced, lymphocytes were eliminated, but neutrophils and macrophages were not.