Cytokine Message and Protein Expression During Lung Granuloma Formation and Resolution Induced by the Mycobacterial Cord Factor Trehalose-6,6′-Dimycolate

Abstract

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.

In Vivo Activity of Released Cell Wall Lipids ofMycobacterium bovisBacillus Calmette-Guérin Is Due Principally to Trehalose Mycolates

“…In our study, the beads were coated in excess of TDM, delivering an estimated 3-4 g to each mouse. Mycobacterium smegmatis-derived TDM, delivered as an oil-in-water emulsion, induces IL-1␤, IL-6, and TNF-␣ expression in a lung granuloma model with no detectable changes in mRNA for IFN-␥, IL-2, IL-4, IL-10, or IL-12 (18). Similarly, in our model, trehalose mycolates did not induce IL-12p70, the T H 1 cytokine, IFN-␥, or the T H 2 cytokines IL-4 or IL-10.…”

“…Cytokines induced by trehalose mycolates contribute to several aspects of tuberculosis. Levels of TNF-␣ induced by infection or TDM correlate with inflammation at the tissue level (18). TNF-␣ induced by TDM after repeated injections also induces cachexia, a prominent clinical feature in end-stage tuberculosis (12).…”

Mycobacterial glycolipid trehalose 6,6′-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes

“…Historically, acute TDM-induced responses to TDM have been well characterized (Behling et al, 1993;Bekierkunst, 1968;Bekierkunst & Yarkoni, 1973;Perez et al, 2000). The lungs of injected mice demonstrate increased production of proinflammatory cytokines, which correlate well with the development of small, focal monocytic granulomas.…”

“…Bone-marrow-derived macrophages (BMM) were prepared as described previously (Indrigo et al, 2002;Perez et al, 2000) with minor modification. Briefly, bone marrow cells were isolated from BALB/c femurs and differentiated at 1610 6 cells ml 21 in McCoy's base containing 10 % heat-inactivated FBS, 10 ng ml 21 recombinant murine GM-CSF (Cell Sciences), 100 mg penicillin G ml 21 and 50 mg gentamicin ml 21 (complete medium) at 37 uC in 5 % CO 2 for 6 days.…”

“…Relative to surface-associated TDM, organisms genetically altered to produce less TDM, or irregular TDM, exhibit reduced growth and survival both in vivo and in vitro (Armitige et al, 2000;Copenhaver et al, 2004;Glickman et al, 2000;Rao et al, 2005). Perhaps TDM's most interesting attribute is its ability to induce cytokine and chemokine production leading to granuloma formation in the lungs of responsive mice (Behling et al, 1993;Bekierkunst, 1968;Guidry et al, 2004;Lima et al, 2001;Perez et al, 2000;Yamagami et al, 2001). In mice, hypersensitive granulomas produced in response to TDM produce pathology which mimics in many ways that seen in M. tuberculosis infection in man.…”