Michael Huber

CD14 is required for MyD88-independent LPS signaling

The recessive mutation Heedless (hdl) was detected in third-generation N-ethyl-N-nitrosourea–mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF–dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4–MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4–MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

Monomeric IgE Stimulates Signaling Pathways in Mast Cells that Lead to Cytokine Production and Cell Survival

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence‐like growth arrest after about 4 wk and shortened postmitotic life span from 1–1/2 years down to 3 months. During the first 8 wk of hyperoxia‐induced ‘aging’, overall protein degradation (breakdown of [35S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin‐specific activity (using the fluorogenic substrate z‐FR‐MCA) and cytoplasmic proteasome‐specific activity (measured with suc‐LLVY‐MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid‐loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.–Sitte, N., Huber, M., Grune, T., Ladhoff, A., Doecke, W.‐D., von Zglinicki, T., Davies, K. J. A. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts. FASEB J. 14, 1490–1498 (2000)

Adenosine and cAMP are potent inhibitors of the NF‐κB pathway downstream of immunoreceptors

Anergic B lymphocytes exert compromised signal transduction towards the activation of NF‐κB in response to B cell antigen receptor (BCR) triggering, whereas activation of the ERK pathway appears normal. How this differential down‐regulation of the NF‐κB pathway is regulated remains still elusive. Here, we demonstrate that stimuli known to enhance 3′,5′‐cyclic adenosine monophosphate (cAMP) are capable of selectively suppressing the activation both of NF‐κB downstream of the BCR and Toll‐like receptor 4 in splenic B lymphocytes and of the high‐affinity receptor for IgE in BM‐derived mast cells. This suppression is accomplished by blocking phosphorylation and subsequent degradation of the inhibitor of NF‐κB. A cAMP‐dependent protein kinase (PKA) inhibitor reverses this suppressive effect, indicating that PKA is a downstream effector of cAMP in this process. Importantly, not only drugs that artificially elevate intracellular cAMP levels, but also the nucleoside adenosine, which is known to be a mediator of cellular distress, inhibit the NF‐κB pathway. This suggests that adenosine‐mediated signals represent an important step in the molecular decision process controlling inflammation versus anergic immune responses.

SHIP Negatively Regulates IgE + Antigen-Induced IL-6 Production in Mast Cells by Inhibiting NF-κB Activity

We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5′-phosphatase (SHIP)−/− than in SHIP+/+ bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP+/+ BMMCs requires the enzymatic activity of SHIP, because SHIP−/− BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP+/+ BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP−/− BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP−/− cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-κB and that IκB phosphorylation/degradation and NF-κB translocation, DNA binding and transactivation are much higher in SHIP−/− BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of IκB and NF-κB DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-κB. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-κB activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.

R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram‐negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S‐ and R‐form LPS from wild‐type and R‐mutants of Salmonella and E. coli, we show that R‐form LPS readily activates mouse cells expressing the signaling receptor Toll‐like receptor 4/myeloid differentiation protein 2 (TLR4/MD‐2), while the S‐form requires further the help of the LPS‐binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R‐form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S‐form LPS. We also show that soluble CD14 at high concentrations enables CD14‐negative cells to respond to S‐form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF‐α levels in response to injection of R‐ and S‐form LPS in mice. Since the R‐form LPS constitutes ubiquitously part of the total LPS present in all wild‐type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

Lipopolysaccharide sensing an important factor in the innate immune response to Gram-negative bacterial infections: Benefits and hazards of LPS hypersensitivity

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.

SHIP Down-Regulates FcεR1-Induced Degranulation at Supraoptimal IgE or Antigen Levels

Cross-linking of the IgE-loaded high-affinity IgE receptor (FcεR1) by multivalent Ags results in mast cell activation and subsequent release of multiple proinflammatory mediators. The dose-response curve for FcεR1-mediated degranulation is bell-shaped, regardless of whether the IgE or the Ag concentration is varied. Although overall calcium influx follows this bell-shaped curve, intracellular calcium release continues to increase at supraoptimal IgE or Ag concentrations. As well, overall calcium mobilization adopts more transient kinetics when stimulations are conducted with supraoptimal instead of optimal Ag concentrations. Moreover, certain early signaling events continue to increase whereas degranulation drops under supraoptimal conditions. We identified SHIP, possibly in association with the FcεR1 β-chain, as a critical negative regulator acting within the inhibitory (supraoptimal) region of the dose-response curve that shifts the kinetics of calcium mobilization from a sustained to a transient response. Consistent with this, we found that degranulation of SHIP-deficient murine bone marrow-derived mast cells was not significantly reduced at supraoptimal Ag levels. A potential mediator of SHIP action, Bruton’s tyrosine kinase, did not seem to play a role within the supraoptimal suppression of degranulation. Interestingly, SHIP was found to colocalize with the actin cytoskeleton (which has been shown previously to mediate the inhibition of degranulation at supraoptimal Ag doses). These results suggest that SHIP, together with other negative regulators, restrains bone marrow-derived mast cell activation at supraoptimal IgE or Ag concentrations in concert with the actin cytoskeleton.

Activation of Murine Macrophages via TLR2 and TLR4 Is Negatively Regulated by a Lyn/PI3K Module and Promoted by SHIP1

Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMMΦs) and in vivo. LPS-stimulated Lyn−/− BMMΦs produced significantly more IL-6, TNF-α, and IFN-α/β compared with wild type (WT) BMMΦs, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMMΦs in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn−/− BMMΦs, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1−/− BMMΦs, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMMΦs. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.

Enhanced B-cell activation mediated by TLR4 and BCR crosstalk

Despite the important role of B lymphocytes as a bridge between the innate and the adaptive immune system, little is known regarding lipopolysaccharide (LPS) recognition, activation of signalling networks or conceivable cooperation between LPS and the B‐cell antigen receptor (BCR). Here, we show that primary B cells can efficiently discriminate between different LPS chemotypes, responding with at least 100‐fold higher sensitivity to rough‐form LPS compared with smooth‐form LPS. Using genetically modified mice, we demonstrate that B lymphocytes recognize all LPS chemotypes via Toll‐like receptor 4 (TLR4). In addition, we dissect the signalling pathways that lead to CD69 upregulation upon TLR4 and BCR activation in primary B cells. Our data suggest that TLR4 and BCR induce CD69 transcription via two distinct sets of signalling molecules, exerting quantitative and qualitative differences in B‐cell activation. Finally, we show that simultaneous stimulation of TLR4 and BCR additively elevates B‐cell activation. In contrast, co‐engagement of TLR4 and BCR by antigen‐coupled LPS synergistically enhances activation of B cells, pointing out attractive targets for signalling crosstalk in B lymphocytes.