Journal of Clinical Immunology, Vol. 23, No. 2, March 2003 ( C  2003) Treatment with an Anti-CD14 Monoclonal Antibody Delays and Inhibits Lipopolysaccharide-Induced Gene Expression in Humans in Vivo C. ARNOLD SPEK, 1,5 ANNELIES VERBON, 2 HELLA ABERSON, 1 JOHN P. PRIBBLE, 3 CATHAL J. MCELGUNN, 4 TERENCE TURNER, 3 TIM AXTELLE, 3 JAN SCHOUTEN, 4 TOM VAN DER POLL, 1,2 and PIETER H. REITSMA 1 Accepted: December 2, 2002 CD14 is a receptor important for activation of cells by lipopolysaccharide (LPS). Treatment with the CD14 antibody IC14 was previously found to attenuate the release of proin- flammatory cytokines and some chemokines into the circulation of healthy humans intravenously injected with LPS. To deter- mine the role of circulating leukocytes in CD14-dependent gene expression, 16 healthy volunteers received LPS preceded by ei- ther IC14 or placebo. At different time points, mRNA was iso- lated from whole blood and gene expression was determined by multiplex ligation-dependent probe amplification (MLPA). LPS induced MIP-1α, MIP-1β , IL-8, IL-1β , and IL-1Ra mRNA pro- duction, which was delayed by 1 hr and reduced twofold by IC14 treatment. TNFR1 was unresponsive, whereas other investigated cytokines remained undetectable. Further, LPS showed differen- tial effects on NFκ B gene expression. LPS induced Iκ Bα pro- duction, whereas p50 was unresponsive and p65 and p49/p100 remained undetectable. LPS induced Iκ Bα expression was de- layed (1 hr) and reduced by IC14. Gene expression profiles in blood cells corresponded poorly with observed changes in plasma levels. These data suggest that peripheral blood cells are of negligible importance in LPS-induced production of inflam- matory mediators in vivo and that LPS may activate genes via a CD14-independent pathway that is slower and less efficient. KEY WORDS: MLPA; CD14; gene expression; endotoxemia and cytokines. 1 Laboratory for Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 2 Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 3 ICOS Corporation, Bothell, Washington. 4 MRC-Holland, Amsterdam, The Netherlands. 5 To whom correspondence should be addressed at Laboratory for Ex- perimental Internal Medicine, G2-131, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax: +31-20- 6977192; e-mail: c.a.spek@amc.uva.nl. INTRODUCTION Lipopolysaccharide (LPS), also called endotoxin, is a component of the outer membrane of Gram-negative bacteria that activates the innate immune system (1–3). Although this activation is important for an adequate host defense against Gram-negative bacteria, under cer- tain conditions the uncontrolled activation of the immune system can result in life-threatening clinical symptoms of sepsis (4). The initial step in cellular activation is the interaction of LPS with LPS binding protein (LBP) (5), with the subsequent transfer of LPS to the cell sur- face receptor CD14 present on different cell types, in- cluding monocytes, macrophages, and granulocytes (6). Alternatively, the LPS–LBP complex interacts with sol- uble CD14 found in plasma, resulting in the activa- tion of CD14-negative cells, as, for instance, endothelial cells (7, 8). Recognition of LPS by CD14 triggers signal transduc- tion through Toll-like receptor 4 (9, 10), eventually leading to activation of nuclear factor (NF)κ B (11) and produc- tion of a series of pro- and antiinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-1 receptor antagonist (IL-1Ra), and macrophage inflammatory proteins (MIP)-1α and -1β (12–14). The CD14 dependence of LPS-induced signaling re- sponses provides a rationale for blocking CD14 function to reduce the consequences of Gram-negative septic shock. Indeed, both animal and human studies have shown that blocking CD14 almost completely inhibits the inflamma- tory response evoked by LPS or Gram-negative bacteria (13, 15–17). In accordance, treatment with the anti-CD14 antibody IC14 during human endotoxemia strongly in- hibited LPS-induced proinflammatory cytokine release, whereas the release of the antiinflammatory cytokines 132 0271-9142/03/0300-0132/0 C  2003 Plenum Publishing Corporation