alveolar macrophages (AMs) after 24 hours of in vitro culture in complete medium

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1 Online Supplement for: NF-κB ACTIVATION IN ALVEOLAR MACROPHAGES REQUIRES IκB KINASE-β, BUT NOT NF-κB INDUCING KINASE Supershift and Competition Assays for NF-κB Competition and supershift assays were performed to confirm that the highlighted bands were nuclear factor-κb (NF-κB). These studies were performed on the nuclear extracts of alveolar macrophages (AMs) after 24 hours of in vitro culture in complete medium (Roswell Park Memorial Institute medium [RPMI] with 5% fetal calf serum [FCS], 25 mm Hepes, 2 mm L-glutamine, and 100 units/ml penicillin/streptomycin). The extracts were prepared according to the protocol described in the main article. The competition assay comprised of three reactions: the first containing 20 µg of protein incubated with [γ- 32 P]-ATP labeled NF-κB alone; the second [γ- 32 P]-ATP labeled NF-κB plus 100 x unlabelled NF-κB; and the third [γ- 32 P]-ATP labeled NF-κB plus a selected nonspecific (AP-1) oligonucleotide (Figure E1). The disappearance of both the upper and lower bands (arrows) confirmed that they were NF-κB specific. The supershift assays involved incubation of the nuclear proteins for two hours at 4 C with antibodies specific to the four known NF-κB components (p50, RelB, c-rel, and p65) before the [γ- 32 P]-ATP labeled NF-κB oligonucleotide was added. This study confirmed that the upper band previously demonstrated to be NF-κB specific contained the p50 and p65 components (Figure E2). This would indicate that the upper NF-κB band was mainly comprised of the classical p50/p65 heterodimer.

2 METHODS Infection of Human Umbilical Cord Vein Endothelial Cells with AdvIKKβkd and AdvIκBα, but Not AdvNIKkd, Inhibits Cytokine Induced IL-6 Production Placental specimens were collected from the Chelsea and Westminster Labour Ward. Human umbilical cord vein endothelial cells (HUVECs) were isolated from umbilical veins by digestion with 0.25-mg/ml collagenase (Clostridium histolyticum; Boehringer, Mannheim, Germany) and propagated in RPMI, containing 10% FCS, 10% newborn calf serum, heparin (90 µg/ml), and endothelial cell growth supplement (20 µg/ml). HUVECs were removed between the second and fifth passage by typsinisation and seeded in wells precoated with gelatin. The cells were then either left uninfected or infected with the stated Adv construct at a multiplicity of infection (m.o.i.) of 150 plaque forming units (pfu):1 before being activated with TNF-α 10 ng/ml or IL-1 10 ng/ml. The mean percentage IL-6 production by cells infected with AdvGFP, AdvNIKkd, AdvIKKβkd, and AdvIκBα was calculated relative to uninfected cells from the same specimen (error bars = standard deviation). Results In a situation analogous to AMs, there was no significant difference in TNF-α and IL-1 mediated IL-6 production by AdvGFP and AdvNIKkd infected HUVECs relative to uninfected cells. There was, however, a substantial reduction in IL-6 production by AdvIKKβkd and AdvIκBα infected HUVECs relative to uninfected cells (Table E1 and Figure E3). This data indicates that primary human cells other than diseased AMs possess

3 a NF-κB inducing kinase (NIK) independent mechanism of cytokine mediated NF-κB activation. Over-Expression of NIKwt Is a Potent Activator of NF-κB Dependent Gene Expression in Alveolar Macrophages Studies performed with various cell lines have reported that over-expression of a wild type (wt) form of NIK is a potent activator of NF-κB dependent gene transcription (E1 E3). To establish if NIKwt was a similarly potent activator of NF-κB dependent gene transcription in primary macrophages, TNF-α production by AMs infected with AdvNIKwt at a progressively greater titre was analyzed by enzyme-linked immunosorbant assay (ELISA) at 24 hours. The incremental induction of cytokine production with the rising titre of infection indicates that over-expression of NIKwt is a potent activator of NF-κB dependent gene transcription. There was no significant alteration in TNF-α production by AMs infected with the AdvGFP control construct at the corresponding titres. This figure is representative of three consecutive experiments performed on different broncoalveolar lavage samples. Even infection of AMs with the AdvNIKwt construct at a titre as low as 30 pfu:1 resulted in a significant induction of proinflammatory cytokine production (Figure E4). IL-6 Production by HUVECs Activated by NIKwt Over-Expression Is Inhibited by Coinfection with AdvNIKkd, AdvIKKβkd, and AdvIκBα To provide further evidence that the NIK encoded by the AdvNIKkd construct was kinase defective (kd), IL-6 production by AMs coinfected with AdvNIKwt and either

4 AdvGFP, AdvNIKkd, AdvIKKβkd, or AdvIκBα was analyzed in a different primary cell lineage that is permissive to Adv infection. Again HUVECs were used for analysis. Untreated HUVECs produce minimal background IL-6, but like AMs demonstrate dosedependent augmentation of IL-6 production following infection with a progressively increasing titre of AdvNIKwt. IL-6 production was analyzed in HUVECs activated with AdvNIKwt (m.o.i.100 pfu:1) following coinfection with either AdvGFP, AdvNIKkd, AdvIKKβkd, or AdvIκBα (Figure E5). The mean and standard deviation of percentage IL-6 production by cells coinfected with the stated Adv vector was calculated relative to cells infected with AdvNIKwt alone. IL-6 production by HUVECs coinfected with AdvNIKwt and AdvNIKkd, AdvIKKβkd or AdvIκBα was reduced to %, % and % respectively (range: 17,638 22,011 pg/ml). References E1. Song HY, Regnier CH, Kirschning CJ, Goeddel DV, Rothe M. Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappab and c-jun N- terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc Natl Acad Sci USA 1997;94: E2. Karin M, Delhase M. JNK or IKK, AP-1 or NF-kappaB, which are the targets for MEK kinase 1 action? Proc Natl Acad Sci USA 1998;95: E3. Shinkura R, Kitada K, Matsuda F, Tashiro K, Ikuta K, Suzuki M, Kogishi K, Serikawa T, Honjo T. Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase. Nat Genet 1999;22:74-77.

5 TABLE E1 TNF-α IL-1 Uninfected % % AdvGFP % % AdvNIKkd % % AdvIKKβkd % % AdvIκBα % %

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