Andreas Kaufmann

Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.

TLR13 Recognizes Bacterial 23S rRNA Devoid of Erythromycin Resistance–Forming Modification

A Double Escapee Toll-like receptors (TLRs)—TLR2 and TLR7—are thought to contribute to the sensing of Gram-positive bacteria like Staphylococcus aureus and Streptococcus pneumoniae by the immune system. Mice deficient in these receptors, however, are still sensitive to infection with these bacteria. Oldenburg et al. (p. 1111, published online 19 July) demonstrate that TLR13 also plays a role in detecting Gram-positive bacteria. TLR13 recognized a conserved region in the peptidyl transferase loop of bacterial 23S ribosomal RNA. Intriguingly, this same sequence is modified by specific methyltransferases that confer resistance to erythromycin. Indeed, erythromycin-resistant bacteria were no longer detectible by TLR13. A region of ribosomal RNA that confers antibiotic resistance is also recognized by mouse innate immune receptors. Host protection from infection relies on the recognition of pathogens by innate pattern-recognition receptors such as Toll-like receptors (TLRs). Here, we show that the orphan receptor TLR13 in mice recognizes a conserved 23S ribosomal RNA (rRNA) sequence that is the binding site of macrolide, lincosamide, and streptogramin group (MLS) antibiotics (including erythromycin) in bacteria. Notably, 23S rRNA from clinical isolates of erythromycin-resistant Staphylococcus aureus and synthetic oligoribonucleotides carrying methylated adenosine or a guanosine mimicking a MLS resistance–causing modification failed to stimulate TLR13. Thus, our results reveal both a natural TLR13 ligand and specific mechanisms of antibiotic resistance as potent bacterial immune evasion strategy, avoiding recognition via TLR13.

Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response.

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4–6 h. Most macrophages died by apoptosis within 25–30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), IL‐6, interferon (IFN) ‐α/β, and CC‐chemokines. The basic mechanisms of virus‐induced cytokine expression are still unknown and appear to involve transcription factors such as nuclear factor‐κB and AP‐1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC‐chemokines macrophage inflammatory protein 1α (MIP‐1α), MIP‐1β, and RANTES were produced while the prototype neutrophil CXC‐chemoattractants IL‐8 and GRO‐α were entirely suppressed. This selective induction of CC‐chemokines may explain the preferential influx of mononuclear leukocytes into virus‐infected tissue. Our data show that monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen‐specific defense. J. Leukoc. Biol. 61: 408–414; 1997.

Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes.

Virulence of the intracellular pathogen Brucella for humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucella organisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO‐α, IL‐8) and CC (MIP‐1α, MIP‐1β, MCP‐1, RANTES) chemokines, as well as pro‐ (IL‐6, TNF‐α) and anti‐inflammatory (IL‐10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucella and its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.

Increase of CCR1 and CCR5 expression and enhanced functional response to MIP-1α during differentiation of human monocytes to macrophages

Chemokines and their receptors regulate migration of leukocytes under normal and inflammatory conditions. In this study, we analyzed the CC chemokine receptor (CCR) expression of monocytes differentiatingin vitro to macrophages. We observed a time‐dependent change of expression and functional responsiveness of CCR1, CCR2, and CCR5 within 48 h. Whereas freshly harvested monocytes were strongly attracted by monocyte chemotactic protein 1 (MCP‐1), a specific ligand for CCR2, only a weak response was observed to macrophage inflammatory protein 1α (MIP‐1α), which binds to CCR1 and CCR5. In striking contrast, differentiated macrophages displayed a strong chemotactic response to MIP‐1α and only a weak response to MCP‐1. These findings were paralleled by intracellular calcium shifts. During the time course of monocyte to macrophage differentiation, mRNA levels and surface expression of CCR2 decreased, whereas that of CCR1 and CCR5 increased. The time‐dependent switch from CCR2 on monocytes to CCR1 and CCR5 on mature macrophages reflects a functional change belonging to the differentiation process of monocytes to macrophages and may form the basis for a differential responsiveness of monocytes and macrophages to distinct sets of chemokines.

Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells

BACKGROUND & AIMS Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. Immigrating leukocytes can potentiate the progression of liver fibrosis by release of fibrogenic mediators and cytotoxic actions. The inducible production of neutrophil chemotactic activities in HSCs was investigated to understand the underlying mechanisms responsible for the attraction of leukocytes in the pathogenesis of liver fibrosis. METHODS Cultured HSCs of different transformation grades and after transformation to myofibroblasts (MFBs) were stimulated with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS), respectively. Induced leukocyte chemotactic activities were evaluated by chemotaxis assays, enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS A transformation grade-dependent differential responsiveness of HSCs and MFBs was observed. TNF-alpha-inducible production of chemotactic mediators increased substantially with advancing transformation. Only transformed MFBs were LPS responsive. Macrophage inflammatory protein 2 was identified as one of the inducible chemokines. CONCLUSIONS The results suggest that chemokines play an important role in the pathogenesis of liver fibrosis. Proinflammatory cytokines can initiate the production of chemotactic activities. The more HSCs are transformed to MFBs, e.g., by chronic injury, the more sensitive the cells become to LPS, which may lead to a vicious circle of enhanced fibrogenic and chemotactic mediator production.

Defense against influenza A virus infection: essential role of the chemokine system.

Monocytes/macrophages are highly susceptible to an infection with influenza A virus. After infection, de novo virus protein synthesis is detectable but rapidly interrupted before completion of the first viral replication cycle. Within 24-48 hours the infected monocytes die by apoptosis. Before cell death, infected monocytes initiate a cell-specific immune response. This includes the transcription and subsequent release of TNF-alpha (tumor necrosis factor alpha), IL-1beta (Interleukin 1beta), IL-6, type I inferferons and CC chemokines. Enhanced cytokine mRNA expression is due to a prolonged mRNA stability and an augmented gene transcription. Activation of transcription factors such as NF-kappaB (nuclear factor kappaB) and AP-1 are involved in activation of cytokine mRNA transcription. Infection of monocytes with influenza A virus induces the selective expression of mononuclear leukocyte attracting chemokines, such as MCP-1 (monocyte chemotactic protein 1), MIP-1alpha (macrophage inflammatory protein 1alpha) and RANTES (regulated upon activation, normal T cell expressed and secreted). In striking contrast, the release of the neutrophil-specific chemokines IL-8 (interleukin 8) and GRO-alpha (growth stimulatory activity alpha) is entirely suppressed. This differentially regulated chemokine expression may explain the mononuclear cell infiltrate characteristic for virus-infected tissue. Thus, infection of monocytes/macrophages with influenza A virus primes for a rapid proinflammatory reaction and induces an enhanced immigration of mononuclear cells into infected tissue. Taken together, these mechanisms may prepare the infected host for a fast and virus-specific immune response.

The 2'-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition.

Bacterial transfer RNA can suppress the immunostimulatory activity of other bacterial tRNAs as a result of the presence of a guanosine modification.

Recognition of nucleic acid and nucleic acid analogs by Toll-like receptors 7, 8 and 9

The mammalian immune system senses pathogens through pattern recognition receptors (PRR) and responds with activation. Toll-like receptors (TLRs) that are expressed on immune and non-immune cells play a critical role in this process. As part of the innate immune response, TLRs lead to cellular activation and cytokine production with subsequent initiation of an adaptive immune response. TLR7-9 recognize single-stranded RNA, nucleoside analogs and single-stranded CpG-DNA, respectively, and their activation initiates the immune response against viruses and bacteria. Furthermore, the stimulation of these TLRs may be exploited for adjuvant therapy, vaccination and anti-tumor responses. However, a role in the generation or perpetuation of autoimmune diseases such as systemic lupus erythematosus (SLE) has also been suggested.

Vaccination strategies for the treatment and prevention of cervical cancer.

Purpose of review Immunotherapy of HPV-induced premalignant anogenital lesions and cervical cancer has made impressive progress. HPV as causative agent is targeted by prophylactic and therapeutic vaccination strategies. Preclinical and clinical studies have shown induction of natural and/or vaccine-induced immune responses. This review will summarize the status of vaccine development and clinical testing published since March 2003. Recent findings For prophylactic vaccines there is first clinical evidence of effectivity (ie, 100% protection from HPV infection and dysplasia by virus-like particle (VLP) vaccine-induced neutralizing antibodies). Also, therapeutic vaccines have entered clinical evaluation. While prophylactic VLP vaccines are immunogenic per se, therapeutic vaccines will need further adjuvants to guide T cell differentiation, expansion, survival, and homing to tumor sites. To enhance clinical outcome of successful T cell induction in patients, the susceptibility of the tumor cells for lysis must be addressed in the future, since tumor immune evasion is a severe problem in cervical cancer. Summary While successful prophylactic HPV vaccines have entered large clinical trials, therapeutic HPV vaccines, in spite of T cell induction, lack clinical responses due to the problem of tumor immune evasion. Adjuvants for systemic and local immune modulation will be mandatory for effective therapy.