1. ¹Mycobacteriology Laboratory Branch, Division of Tuberculosis Elimination, Centers for Disease Control and Prevention, Atlanta, GA, USA
2. ²Infectious Disease Pathology Activity, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
3. ³Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
4. ⁴Biotechnology Core Facility Branch, Scientific Resources Program, Centers for Disease Control and Prevention, Atlanta, GA, USA

Correspondence: Dr F Quinn, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, 501 Brooks Drive, Athens, GA 30602, USA. E-mail: fquinn@vet.uga.edu

Received 24 April 2006; Revised 20 July 2006; Accepted 24 July 2006; Published online 2 January 2007.

Abstract

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a person's lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test-negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage-specific CD68⁺ epithelioid macrophages and small round CD3⁺ lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid-fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead-based assay using the Luminex 100 and were found to contain interleukin (IL)-6, IL-8, interferon- and tumor necrosis factor-, cytokines known to be involved in human granuloma formation, in quantities from two-fold to 7000-fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.