Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-α secretion by bovine alveolar macrophages in vitro

Abstract

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils

“…The ability of serum to enhance the responsiveness of leukocytes from other species to LPS [32,33,41], reflects the formation of a complex between LPS and a serum LBP that is recognized by the glycosylphosphatidylinositol-anchored membrane protein, CD14 [42]. These data provide strong evidence for a similar paradigm underlying the mechanism of LPS interaction with equine neutrophils.…”

Binding of Toxoplasma gondii Glycosylphosphatidylinositols to Galectin-3 Is Required for Their Recognition by Macrophages

“…Free GPIs are present in the extracellular medium in their entire form, probably secreted by the parasites. 6 We assume that macrophage enzymes like phospholipases, cleave the GPI lipid moiety, allowing its interaction with galectin-3 and the TLR signaling. Galectin-3 may play a major role in controlling cytokine production.…”

“…We have shown that both TLR2 and TLR4 are involved in the NF-Bdependent signaling cascade leading to production of TNF-␣ by macrophages exposed to T. gondii GPIs (5). The membrane protein CD14 is a co-receptor that associates with TLR4 to form a receptor complex for bacterial lipopolysaccharide (LPS) (6). CD14 is involved in the recognition of lipoproteins from Mycobacterium tuberculosis by TLR2 (7).…”

Recognition of Staphylococcus aureus-derived peptidoglycan (PGN) but not intact bacteria is mediated by CD14 in microglia

“…Since microglia represent the first line of defense against bacterial infections in the CNS parenchyma and modulating PRR expression may have implications on the extent of microglial antibacterial responses, the effects of S. aureus and PGN on CD14 protein expression in primary microglia was evaluated by Western blotting. Since serum contains sCD14 and LBP that are capable of augmenting cell responses to microbial stimuli (Cohen et al, 1995;Wright et al, 1990;Yang et al, 1994), the ability of S. aureus and PGN to modulate CD14 expression was evaluated in the absence of serum. Under these conditions, both S. aureus and PGN stimulation led to an increase in CD14 protein expression in primary WT microglia (Fig.…”

“…In the current study, we evaluated the relative importance of CD14 on S. aureus and PGN recognition using primary microglia isolated from CD14 KO and WT mice. Since fetal bovine serum (FBS) serves as a source of sCD14 and LPS binding protein (LBP), the latter of which facilitates the transfer of PGN to mCD14 (Cohen et al, 1995;Dziarski, 2003;Wright et al, 1990;Yang et al, 1994), we examined microglial activation in response to gram-positive stimuli under serum-free conditions to prevent any confounding effects of serum on the results obtained. The findings presented demonstrate that CD14 participates in PGN-dependent microglial activation, whereas responses to intact S. aureus are primarily CD14-independent.…”