IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock- in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1 beta or a Toll- like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NF kappa B, JNK, and p38 activation in response to IL-1 beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1 beta response genes and revealed that the induction of IL-1 beta-responsive mRNAs is largely ablated in IRAK-4KDcells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.

The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1 beta induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1 beta induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases' transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium, A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.

Most cases of early onset familial Alzheimer's disease (FAD) involve mutations in presenilins (PS1 and PS2) genes. The C-terminal portion of PS2 is a homologue with an apoptosis-linked gene (ALG-3). To characterise the role of PS1 in apoptosis, we overexpressed the corresponding C-terminal fragment of PS1 (PS1-f) under the control of the tetracycline-responsive transactivator in Jurkat cells. The tight regulation of the expression of the 11 kDa PS1-f peptide was verified. A 50% inhibition of anti-Fas induced apoptosis was observed upon PS1-f transient overexpression compared to the repressed state. Stable transfectants selectively overexpressing PS1-f revealed a transient protective effect of 30% after apoptosis induction.

A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established, Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*) containing peptide library, 6.4 x 10(5) individual peptides of nine amino acids in length (EPX(6)Y*X(19)X(7)X(19)X(7)X(6)) were each displayed on beads, Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting, Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk, selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine, For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.

A transgenic mouse system has been established to follow the pattern of IL-2 expression at the level of single T cells. This was achieved by introducing a human IL-2 promoter-driven reporter gene (Escherichia coli lacZ) into the germline of mice and monitoring its product, beta-galactosidase (beta-gal), by FACS analysis. Ex vivo experiments confirmed that the regulated expression of the transgene is comparable with that of the endogenous IL-2 gene. Transgene expression is inducible by mitogens, restricted to T cells, and diminished by immunosuppressive agents, such as cyclosporin A, at concentrations known to suppress IL-2 transcription. Depending on the mitogens used, 30-50% of peripheral T cells produced IL-2 with an asynchronous induction pattern, as measured by transgenic beta-gal activity. Both helper (CD4+CD8-) and cytotoxic T cells (CD4-CD8+) respond with comparable heterogenous expression levels but they show different frequencies of beta-gal production. Transgenic beta-gal-producing T cells were detectable as early as 2 h after mitogen stimulation. These cells represent a transitional IL-2 secreting, IL-2 receptor alpha-chain negative T cell population, which occurs in the autocrine process of T cell activation. Administration of staphylococcal enterotoxin A (SEA), a bacterial superantigen, resulted in a T cell specific (Thy-1.2) increase (2.5-fold) of reporter gene expression in vivo. In summary, we could demonstrate that IL-2 promoter-driven reporter gene expression in transgenic mice is a sensitive tool to characterize IL-2 expressing cells phenotypically.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-beta but only weakly to Ig-alpha. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-alpha/beta heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-alpha and Ig-beta might couple to different biochemical pathways.