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Infection and Immunity, January 2000, p. 257-263, Vol. 68, No. 1
0019-9567/0/$04.00+0

Effects of Opsonization and Gamma Interferon on Growth of Brucella melitensis 16M in Mouse Peritoneal Macrophages In Vitro

Michael O. Eze,1,dagger Liang Yuan,1 Robert M. Crawford,2,Dagger Chrysanthi M. Paranavitana,1 Ted L. Hadfield,3 Apurba K. Bhattacharjee,1 Richard L. Warren,1,§ and David L. Hoover1,*

Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100,1 and American Registry of Pathology2 and Department of Infectious and Parasitic Diseases,3 Armed Forces Institute of Pathology, Washington, D.C. 20306-6000

Received 10 June 1999/Returned for modification 16 July 1999/Accepted 20 October 1999

Entry of opsonized pathogens into phagocytes may benefit or, paradoxically, harm the host. Opsonization may trigger antimicrobial mechanisms such as reactive oxygen or nitric oxide (NO) production but may also provide a safe haven for intracellular replication. Brucellae are natural intramacrophage pathogens of rodents, ruminants, dogs, marine mammals, and humans. We evaluated the role of opsonins in Brucella-macrophage interactions by challenging cultured murine peritoneal macrophages with Brucella melitensis 16M treated with complement- and/or antibody-rich serum. Mouse serum rich in antibody against Brucella lipopolysaccharide (LPS) (aLPS) and human complement-rich serum (HCS) each enhanced the macrophage uptake of brucellae. Combinations of suboptimal levels of aLPS (0.01%) and HCS (2%) synergistically enhanced uptake. The intracellular fate of ingested bacteria was evaluated with an optimal concentration of gentamicin (2 µg/ml) to control extracellular growth but not kill intracellular bacteria. Bacteria opsonized with aLPS and/or HCS grew equally well inside macrophages in the absence of gamma interferon (IFN-gamma ). Macrophage activation with IFN-gamma inhibited replication of both opsonized and nonopsonized brucellae but was less effective in inhibiting replication of nonopsonized bacteria. IFN-gamma treatment of macrophages with opsonized or nonopsonized bacteria enhanced NO production, which was blocked by NG-monomethyl L-arginine (MMLA), an NO synthesis inhibitor. MMLA also partially blocked IFN-gamma -mediated bacterial growth inhibition. These studies suggest that primary murine macrophages have limited ability to control infection with B. melitensis, even when activated by IFN-gamma in the presence of highly opsonic concentrations of antibody and complement. Additional cellular immune responses, e.g., those mediated by cytotoxic T cells, may play more important roles in the control of murine brucellosis.

* Corresponding author. Mailing address: Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100. Phone: (301) 319-9573. Fax: (301) 319-9123. E-mail: david.hoover{at}na.amedd.army.mil.

dagger Present address: Department of Chemistry, University of Winnipeg, Winnipeg, Manitoba, Canada R3B2E9.

Dagger Present address: Hemagen Diagnostics, Inc., Columbia, MD 21045.

§ Present address: Dugway Proving Grounds, Dugway, Utah.

Infection and Immunity, January 2000, p. 257-263, Vol. 68, No. 1
0019-9567/0/$04.00+0


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