Direct Evidence for Involvement of NF-κB in Transcriptional Activation of Tumor Necrosis Factor by a Spirochetal Lipoprotein

Abstract

Variable major lipoprotein (Vmp) is a major tumor necrosis factor (TNF)-inducing component of Borrelia recurrentis, the agent of louse-borne relapsing fever. B. recurrentis Vmp rapidly stimulates nuclear translocation of NF-κB and proinflammatory cytokine gene expression in the human monocyte-like cell line MonoMac 6. By overexpressing disabled mutant IκBα in MonoMac 6 cells cotransfected with a reporter gene, we provide evidence that NF-κB is essential for the transcriptional activation of TNF in this system.

Louse-borne relapsing fever (LBRF) caused by the spirochete Borrelia recurrentis provides one of the clearest examples of the causal role of tumor necrosis factor (TNF) in the pathogenesis of fever in humans. When patients with LBRF are treated with antibiotics such as penicillin, a significant proportion develop an exacerbation of fever and other clinical symptoms. This phenomenon, known as the Jarisch-Herxheimer (J-H) reaction (12), can be suppressed by administration of anti-TNF antibody prior to antibiotic therapy (3). Using a biochemical purification strategy, we previously identified a major TNF-inducing factor of B. recurrentis to be a variable major lipoprotein (Vmp) that has significant homology to the Vmps identified in B. hermsii and other borrelial species (16). The TNF-inducing activity resides in the lipid moiety which has been characterized in some detail (13). Thus, a plausible explanation of the J-H reaction is that cell lysis caused by antibiotic therapy results in the exposure of proinflammatory lipid structures that are normally embedded within the spirochetal cell membrane.

Interaction of spirochete surface lipoproteins with the Toll-like receptor 2 (TLR 2) present on macrophages has been proposed as the critical initial contact between host and pathogen (5). Occupancy of TLR leads to activation of a signaling cascade that results in translocation of the transcription factor complex NF-κB into the nucleus (9). Since the promoter regions of TNF and other proinflammatory cytokine genes contain consensus binding sequences for NF-κB, it has been proposed that this transcription factor acts to initiate the host response to spirochetal infection (8). In human endothelial cells, B. burgdorferi outer surface lipoprotein A has been shown to induce NF-κB nuclear translocation (19), and proinflammatory cytokine release can be suppressed by chemical inhibitors of NF-κB (2). The goal of this study was to obtain direct evidence that NF-κB plays a causal role in the transcriptional activation of TNF by the Vmp of B. recurrentis.

Kinetics of proinflammatory cytokine mRNA expression induced by VmpA.

We investigated the synthesis of TNF, interleukin-1β (IL-1β) and IL-12 p40 mRNA by a human monocyte-like cell line (MonoMac 6) following stimulation with Vmp isolated from clinical isolate A1 (VmpA1) or a known monocyte activator, Escherichia coli O55:B5 lipopolysaccharide (LPS). LPS-free VmpA1 was purified as described elsewhere (16). Preliminary dose-response experiments revealed that similar amounts of TNF were secreted by MonoMac 6 cells stimulated with VmpA1 (300 ng/ml; equivalent to 8.5 nM, as the molecular mass of VmpA1 is 35,077 Da as determined by mass spectrometry [13]) or E. coli O55:B5 LPS (10 ng/ml; equivalent to 2 nM, assuming an average molecular mass of 5,000 Da), and these concentrations were used in this study. MonoMac 6 cells were stimulated for various lengths of time, and levels of messages were quantified by Northern analysis (Fig. 1). Total RNA was prepared using Tri reagent (Sigma). Ten micrograms of RNA was separated on a 1.4% agarose–1.8 M formaldehyde gel and transferred to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech). The membrane was hybridized with either ³²P-labeled human TNF, IL-1β, or IL-12 p40 cDNA fragments that were generated by reverse transcription-PCR using primers from Stratagene and RNA from MonoMac 6 cells stimulated with LPS. Signal intensities for each cDNA probe were compared to the level of expression of β-actin mRNA. TNF mRNA was not detected in untreated cells, but levels increased rapidly after stimulation with VmpA1 or LPS. In both cases TNF mRNA was detectable at 30 min, became maximal at 2 h, and was greatly reduced by 4 h. Message for IL-1β was detected within 2 h of stimulation with VmpA1 or LPS, remained maximal for up to 8 h, and declined to undetectable levels within 24 h. Similar results were obtained for IL-12 p40 mRNA. These results indicate that stimulation of MonoMac 6 cells with VmpA1 or LPS leads to similar expression profiles of proinflammatory mediators. In addition, the rapid and transient induction of TNF expression in response to VmpA1 in vitro is consistent with the clinical data showing a rapid increase in this pyrogen preceding the onset of the J-H reaction (7).

FIG. 1.

Northern analysis of cytokine induction by VmpA1 and LPS. mRNA was isolated from MonoMac 6 cells that were stimulated for the times shown with LPS (10 ng/ml; 2 nM) or VmpA1 (300 ng/ml; 8.5 nM). Following electrophoresis, mRNA was transferred to nitrocellulose, and the blots were hybridized with TNF, IL-1β, IL-12 p40, and actin cDNA probes as shown.

Induction of NF-κB translocation by VmpA1.

The rapidity of the rise in TNF mRNA level suggests that VmpA1-mediated cell activation does not require protein synthesis de novo but recruits preexisting transcription factors. We have shown previously that LPS stimulation of MonoMac 6 cells results in rapid nuclear translocation of NF-κB/Rel transcription factors (15). The similarity in activation kinetics between VmpA1 and LPS led us to investigate whether VmpA1 also activates this family of transcription factors. Labeled oligoduplexes corresponding to a NF-κB site derived from the human immunodeficiency virus (HIV) long terminal repeat were added to nuclear extracts of MonoMac 6 cells that had been stimulated with VmpA1 for various lengths of time and were subjected to electrophoretic mobility shift assay (EMSA) (Fig. 2A). Nuclear protein extraction, binding reaction, and electrophoresis were performed exactly as previously described (15). Translocation of NF-κB to the nucleus reached a plateau within 30 min of stimulation by VmpA1 (lanes 7 to 12) or LPS (lanes 1 to 6) and declined after 2 h of treatment. Addition of antibodies directed against subunit p50 or p65 of the NF-κB complex resulted in the formation of slower-migrating complexes, confirming that these factors are bound to the oligoduplex (Fig. 2B). VmpA1 induces occupancy of three κB-binding sites from the human TNF promoter (sites κB2, κB2a, and ξ [Fig. 2C]) as well as a κB-binding site from the downstream regulatory region (site κB4). These results indicate that VmpA1 rapidly induces NF-κB translocation in MonoMac 6 cells. Moreover, induced NF-κB complexes are competent to bind κB sites derived from regulatory regions of the human TNF gene, suggesting that up-regulation of the TNF gene by VmpA1 may be mediated by NF-κB activation.

FIG. 2.

(A) EMSA using an HIV NF-κB oligoduplex radiolabeled with [α-³²P]dCTP (Amersham Pharmacia Biotech) (forward [F], 5′-agctCTACAAGGGACTTTCCGCTGGG; reverse [R], 5′-agctCCCAGCGGAAAGTCCCTTGTAG) and nuclear extracts prepared from MonoMac 6 cells stimulated for the times shown with LPS (10 ng/ml) or VmpA1 (300 ng/ml). (B) Supershift analysis of complexes formed using radiolabeled HIV NF-κB oligoduplex and a nuclear extract from MonoMac 6 cells stimulated for 1 h with LPS (10 ng/ml) or VmpA1 (300 ng/ml). Complexes were incubated without (−) or with (+) anti-p50, anti-p65, and antibiotin (an irrelevant antibody) antibodies (Abs) as shown. (C) EMSA using radiolabeled NF-κB oligoduplexes corresponding to sequences within the TNF promoter (κB2 F [5′-agctGGGTCTGTGAATTCCCGGGGGT] and R [5′-agctACCCCCGGGAATTCACAGACCC]; κB2a F [5′-agctTCCCCGGGGCTGTCCCAGGCTT] and R [5′-agctAAGCCTGGGACAGCCCCGGGGA]; κB4 F [5′-agctGGGCATGGGAATTTCCAACTCT] and R [5′-agctAGAGTTGGAAATTCCCATGCCC]) and nuclear extracts prepared from MonoMac 6 cells stimulated for 1 h with VmpA1 (300 ng/ml).

NF-κB is involved directly in TNF gene activation by VmpA1.

To investigate directly the role of NF-κB in TNF mRNA expression following VmpA1 stimulation, MonoMac 6 cells that are defective in translocation of NF-κB into the nucleus were used. These cells were generated either by overexpression of IκB or by creation of a nondegradable IκB mutant (18). In previous studies we have shown that MonoMac 6 cells transiently transfected with a luciferase reporter gene driven by the full-length human TNF promoter express luciferase activity following stimulation with LPS (15). In this study we cotransfected MonoMac 6 cells with a construct overexpressing IκB (gift from A. Baldwin, University of North Carolina, Chapel Hill) and the luciferase gene reporter construct. MonoMac 6 cells were transfected using Effectene reagent (Qiagen) according to the manufacturer's instructions. Twenty-four hours posttransfection, cells were stimulated with LPS or VmpA1 for a further 6 h before harvesting. Luciferase activity was then measured according to the Promega protocol. Stimulation of these cells with VmpA1 resulted in a 60% decrease in the level of inducible luciferase activity compared to control cells (Fig. 3). Overexpression of the degradation-deficient IκBa S32/36A mutant (18) resulted in an even greater reduction (80% decrease of the original induced level). From these results, we conclude that NF-κB complexes have an essential role in up-regulation of transcription of the TNF gene in MonoMac 6 cells stimulated with VmpA1.

FIG. 3.

Luciferase activity expressed by MonoMac 6 cells cotransfected with a luciferase reporter gene and either pRcCMV (vector only), pRcCMV-IκB, or pRcCM-^32/36S IκB following stimulation with (10 ng/ml) LPS or VmpA1 (300 ng/ml). Luciferase activities (average of three experiments ± standard deviation) are expressed relative to that of pRcCMV cotransfected MonoMac 6 cells stimulated with LPS.

The pathogenesis of borrelial relapsing fever is a problem of some practical importance, particularly in the tropics. Many parts of Africa have a high incidence of tick-borne relapsing fever, and epidemics of potentially fatal LBRF are prone to occur when populations are displaced (1). Several investigations have demonstrated the proinflammatory properties of membrane lipoproteins expressed by borrelial species and other spirochetes (6, 10, 11, 14, 17), and it has recently been demonstrated that these lipoproteins can stimulate macrophages through TLR 2 (5).

Here we focus on VmpA1, a variable major lipoprotein which we have previously shown to be the major TNF-stimulating component of clinical isolate A1 of B. recurrentis, the agent of LBRF (16). In the human monocyte-like cell line MonoMac 6, we find that TNF mRNA is synthesized within 30 min and disappears within 4 h of stimulation with VmpA1. mRNA for IL-1β and IL-12 p40, other proinflammatory cytokines, appears later (2 h), and levels remain elevated for longer (8 h). These findings are consistent with the clinical observation that TNF is the first cytokine to appear in the circulation following antibiotic treatment of LBRF is TNF (7).

Several studies have implicated the transcription factor NF-κB in the TNF response to borrelial infection (8, 19), but there is little direct evidence that it plays a causal role. The latter question is of interest because whereas NF-κB is known to be of importance in murine TNF regulation, its role as a transcriptional activator of human TNF has been the subject of considerable debate (4, 15). We report two lines of evidence that address this issue. Our kinetic EMSA data demonstrate that nuclear translocation of NF-κB occurs in a human monocyte line within 30 min of stimulation with VmpA1, consistent with the hypothesis that NF-κB is at a proximal part of the proinflammatory pathway. More direct evidence comes from our experiments using a nondegradable mutant of IκB (a dominant negative mutant) and constructs that overexpress IκB, cotransfected into MonoMac 6 cells with a luciferase gene reporter linked to the TNF promoter. We find that overexpression of IκB, or a nondegradable mutant, results in 60 to 80% decrease in luciferase activity induced by VmpA1. These data provide evidence that NF-κB plays a causal role in human cytokine production induced by spirochetal lipoproteins.

The innate inflammatory response may play a significant role in host defense as well as contributing to the clinical symptomatology and pathogenesis of spirochetal infection. Understanding the molecular basis of this response, and how it is regulated, may provide important insights into the development of novel strategies for these treatment or prevention of these globally distributed diseases.