Involvement of CD14 and β2-Integrins in Activating Cells with Soluble and Particulate Lipopolysaccharides and Mannuronic Acid Polymers

Abstract

Lipopolysaccharide (LPS), a glycolipid present in the outer membrane of gram-negative bacteria, is a potent inducer of proinflammatory responses from cells of the monocytic lineage. LPS stimulation of monocytes results in cytokine production, one of the key events in the pathogenesis of gram-negative sepsis (4). The cell surface glycoprotein CD14 (membrane CD14 [mCD14]) has been identified as the principal LPS receptor on phagocytic leukocytes, enabeling them to be stimulated with picogram amounts of LPS (42,47). This process is facilitated by the catalytic activity of the blood protein LPS binding protein (LBP), which accelerates the binding of LPS to mCD14 (20). CD14 exists in two forms; in myeloid cells it is expressed as a glycosylphosphatidylinositol (GPI)-anchored glycoprotein (21), whereas a soluble form of CD14 (sCD14) lacking a GPI tail is present in blood (2). We have previously reported that uronic acid polymers with a ␤ (134) glycosidic linkage are able to stimulate monocytes to produce tumor necrosis factor (TNF) in an mCD14-dependent manner; polymers of high mannuronic acid content (M-polymers) were found to be the most potent (14). Several reports subsequently implicated CD14 in responses to a variety of bacterial compounds (36,37,44), suggesting that the role of CD14 is not limited to LPS recognition.In addition to CD14, other proteins described as LPS receptors include the ␤2-integrins CR3 (CD11b/CD18, Mac-1) and CR4 (CD11c/CD18, p150,95) (46). Wright and coworkers reported that CR3 and CR4 function in the recognition of Escherichia coli by binding to the lipid A portion of LPS (46). However, cells from patients genetically deficient in CD18 expression responded normally to LPS (45), suggesting that CD18 is not essential for cellular responses to LPS. On the other hand, Ingalls et al. found that Chinese hamster ovary (CHO)-K1 cells transfected with CR3 or CR4 acquire LPS responsiveness, as evidenced by inducible NF-B translocation (24,25). Furthermore, components from group B streptococcus (GBS) type III can activate human monocytes to TNF production through a CD18-dependent mechanism (8, 31), suggesting that under certain defined conditions, engagement of the ␤2-integrins by bacterial ligands is proinflammatory. Previously we have reported that covalently linking detoxified LPS (DLPS) and M-polymers to particles increased their TNF-inducing potency 2,000 to 60,000 times compared to that of the polymers in soluble form (3). In the present work we have investigated the mechanisms by which soluble LPS, DLPS, and M-polymers (350 kDa) stimulate monocytes to produce TNF compared to DLPS covalently attached to particles (DLPS-particles) or M-polymers (ϳ3 kDa) covalently attached to particles (M-particles). The data suggest that phagocytes utilize membrane CD14 for LPS-, DLPS-, and Mpolymer-induced TNF production, both in solution and attached to particles. In contrast, the ␤2-integrin CR3 only participates in the response to the particulate form of the polymers. These data suggest that different membr...

Src Tyrosine Kinases Mediate Activations of NF-κB and Integrin Signal during Lipopolysaccharide-Induced Acute Lung Injury

“…Several previous studies have suggested that integrins participate in LPS signaling in macrophages (13,42,43). Furthermore, the binding of ligands to integrin ␣ v ␤ 3 was found to induce in NF-B activation (35), thus suggesting that integrin ␣ v ␤ 3 engagement influences an NF-B-dependent program of gene expression.…”

Involvement of Toll-like Receptor (TLR) 2 and TLR4 in Cell Activation by Mannuronic Acid Polymers

“…Reagents-Poly-M (ϳ350 kDa, 92% M) was isolated from mucoid P. aeruginosa, and the batch was the same as that previously used (4,5 5 monocytes/well in 24-well dishes) were washed three times in Hanks' balanced salt solution (Invitrogen) and pretreated with a 10 g/ml concentration of the indicated mAbs or with 1 g/ml synthetic lipid IVA (compound 406) for 30 min at room temperature in serum-free RPMI 1640 medium (Invitrogen) with 0.01% L-glutamine, 40 g/ml gentamycin (referred to as RPMI hereafter) prior to addition of stimuli. Stimulation proceeded for 8 h at 37°C before supernatants were collected and stored at Ϫ20°C until assayed for TNF activity in the WEHI 164 clone 13 bioassay as described previously (27).…”

“…3). Third, the TNFinducing capabilities of high molecular poly-M is greatly reduced by hydrolytic or enzymatic breakdown but restored or even enhanced when the resultant low molecular chains are attached to particles (4,5). Finally, the lack of response to poly-M in TLR4Ϫ/Ϫ cells rule out possible contamination with lipoproteins or lipopeptides.…”

“…The potency increases with the content of M residues and molecular size, and polymers isolated from mucoid pseudomonads (poly-M, 92-96% M) are the most active (3). However, attaching oligomeric M blocks of low activity to particles enhances their TNFinducing potency by 2-4 log units, suggesting that optimal immunostimulating activity is determined from the polymer conformation (4,5). Given prophylactically, poly-M has also been shown to protect mice from lethal Escherichia coli infection 2 and x-irradiation (6) and to stimulate murine hematopoiesis (6).…”

“…Like lipopolysaccharide (LPS) from Gram-negative bacteria (7), poly-M induces TNF production from human monocytes by binding to the surface receptor CD14 (8), and the response is enhanced by serum as a source of LPS-binding protein and soluble CD14 (5,7,9,10). CD14 lacks an intracellular part (11) and probably mediates cell activation by interaction with other signal transducing molecules.…”