Richard J. Ulevitch

Toll-like receptors in the induction of the innate immune response.

The innate immune response is the first line of defence against infectious disease. The principal challenge for the host is to detect the pathogen and mount a rapid defensive response. A group of proteins that comprise the Toll or Toll-like family of receptors perform this role in vertebrate and invertebrate organisms. This reflects a remarkable conservation of function and it is therefore not surprising that studies of the mechanism by which they act has revealed new and important insights into host defence.

INDEPENDENT HUMAN MAP KINASE SIGNAL-TRANSDUCTION PATHWAYS DEFINED BY MEK AND MKK ISOFORMS

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

Identity of tumour necrosis factor and the macrophage-secreted factor cachectin

In mammals, several well-defined metabolic changes occur during infection, many of which are attributable to products of the reticuloendothelial system1–3. Among these changes, a hypertrigly-ceridaemic state is frequently evident4–9, resulting from defective triglyceride clearance, caused by systemic suppression of the enzyme lipoprotein lipase (LPL)9. We have found previously that macrophages secrete the hormone cachectin, which specifically suppresses LPL activity in cultured adipocytes (3T3-L1 cells)10–17. When originally purified from RAW 264.7 (mouse macrophage) cells, cachectin was shown to have a pI of 4.7, a subunit size of relative molecular mass (Mr) 17,000 and to form non-covalent multimers17. A receptor for cachectin was identified on non-tumorigenic cultured cells and on normal mouse liver membranes17. A new high-yield purification technique has enabled us to determine further details of the structure of mouse cachectin. We now report that a high degree of homology exists between the N-terminal sequence of mouse cachectin and the N-terminal sequence recently determined for human tumour necrosis factor (TNF)18,19. Purified cachectin also possesses potent TNF activity in vitro. These findings suggest that the ‘cachectin’ and ‘TNF’ activities of murine macrophage conditioned medium are attributable to a single protein, which modulates the metabolic activities of normal as well as neoplastic cells through interaction with specific high-affinity receptors.

Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1.

The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-activated kinase 1 (Pak1) is a potential mediator of Rac/Cdc42 signaling, and we observe that Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK.

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Toll-like receptor 2–mediated NF-κB activation requires a Rac1-dependent pathway

Mammalian Toll-like receptors (TLRs) are expressed on innate immune cells and respond to the membrane components of Gram-positive or Gram-negative bacteria. When activated, they convey signals to transcription factors that orchestrate the inflammatory response. However, the intracellular signaling events following TLR activation are largely unknown. Here we show that TLR2 stimulation by Staphylococcus aureus induces a fast and transient activation of the Rho GTPases Rac1 and Cdc42 in the human monocytic cell line THP-1 and in 293 cells expressing TLR2. Dominant-negative Rac1N17, but not dominant-negative Cdc42N17, block nuclear factor-κB (NF-κB) transactivation. S. aureus stimulation causes the recruitment of active Rac1 and phosphatidylinositol-3 kinase (PI3K) to the TLR2 cytosolic domain. Tyrosine phosphorylation of TLR2 is required for assembly of a multiprotein complex that is necessary for subsequent NF-κB transcriptional activity. A signaling cascade composed of Rac1, PI3K and Akt targets nuclear p65 transactivation independently of IκBα degradation. Thus Rac1 controls a second, IκB–independent, pathway to NF-κB activation and is essential in innate immune cell signaling via TLR2.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Participation of tumor necrosis factor in the mediation of gram negative bacterial lipopolysaccharide-induced injury in rabbits.

Macrophages are induced by LPS to release a number of products that determine the host response during gram negative sepsis. To examine the role of one such substance, tumor necrosis factor (TNF), in mediating LPS-induced injury, we employed a rabbit model of endotoxic shock to (a) determine the kinetics and extent of release of TNF into plasma after injection of LPS, and (b) to evaluate the protective effect of in vivo neutralization of LPS-induced TNF by prior infusion of anti-TNF antibody. TNF was maximally induced 45-100 min after injection of 10 micrograms i.v. parent Salmonella minnesota Re595 LPS or 250 micrograms Re595 LPS-HDL complexes. Maximal induction of TNF by LPS was associated with development of hypotension, focal hepatic necrosis, intravascular fibrin deposition and lethality. Based on (a) the peak levels of TNF observed in serum, 2.5 X 10(3) U/ml, (b) the specific activity of purified rabbit macrophage-derived TNF, 1 X 10(8) U/mg, and (c) the biphasic disappearance of intravenously injected purified TNF (t1/2 = 0.5 min, 11 min) we constructed a kinetic model showing that at least 130 micrograms of TNF (1.3 X 10(7) U) was released into plasma 30-200 min postinjection of LPS. Prior infusion of anti-TNF antibody (30-45 min before LPS injection) resulted in neutralization of the LPS-induced serum TNF activity and provided significant protection from the development of hypotension, fibrin deposition, and lethality. Thus, these results provide further evidence that TNF plays a central role mediating the pathophysiologic changes that occur during gram negative endotoxic shock.

BMK1/ERK5 regulates serum-induced early gene expression through transcription factor MEF2C.

Big MAP kinase 1 (BMK1), also known as ERK5, is a mitogen‐activated protein (MAP) kinase member whose biological role is largely undefined. We have shown previously that the activity of BMK1 in rat smooth muscle cells is up‐regulated by oxidants. Here, we describe a constitutively active form of the MAP kinase kinase, MEK5(D), which selectively activates BMK1 but not other MAP kinases in vivo. Through utilization of MEK5(D), we have determined that a member of the MEF2 transcription factor family, MEF2C, is a protein substrate of BMK1. BMK1 dramatically enhances the transactivation activity of MEF2C by phosphorylating a serine residue at amino acid position 387 in this transcription factor. Serum is also a potent stimulator of BMK1‐induced MEF2C phosphorylation, since a dominant‐negative form of BMK1 specifically inhibits serum‐induced activation of MEF2C. One consequence of MEF2C activation is increased transcription of the c‐jun gene. Taken together, these results strongly suggest that in some cell types the MEK5/BMK1 MAP kinase signaling pathway regulates serum‐induced early gene expression through the transcription factor MEF2C.

How we detect microbes and respond to them: the Toll-like receptors and their transducers

Macrophages and dendritic cells are in the front line of host defense. When they sense host invasion, they produce cytokines that alert other innate immune cells and also abet the development of an adaptive immune response. Although lipolysaccharide (LPS), peptidoglycan, unmethylated DNA, and other microbial products were long known to be the primary targets of innate immune recognition, there was puzzlement as to how each molecule triggered a response. It is now known that the Toll‐like receptors (TLRs) are the principal signaling molecules through which mammals sense infection. Each TLR recognizes a restricted subset of molecules produced by microbes, and in some circumstances, only a single type of molecule is sensed (e.g., only LPS is sensed by TLR4). TLRs direct the activation of immune cells near to and far from the site of infection, mobilizing the comparatively vast immune resources of the host to confine and defeat an invasive organism before it has become widespread. The biochemical details of TLR signaling have been analyzed through forward and reverse genetic methods, and full elucidation of the molecular interactions that transpire within the first minutes following contact between host and pathogen will soon be at hand.