Toll-like receptor (TLR)2 and TLR4 agonists regulate CCR expression in human monocytic cells

Interactions between proinflammatory and cell maturation signals, and the pathways that regulate leukocyte migration, are of fundamental importance in controlling trafficking and recruitment of leukocytes during the processes of innate and adaptive immunity. We have investigated the molecular mechanisms by which selective Toll-like receptor (TLR)2 and TLR4 agonists regulate expression of CCR1 and CCR2 on primary human monocytes and THP-1 cells, a human monocytic cell line. We found that activation of either TLR2 (by Pam(3)CysSerLys(4)) or TLR4 (by purified LPS) resulted in down-modulation of both CCR1 and CCR2. Further investigation of TLR-induced down-modulation of CCR1 revealed differences in the signaling pathways activated, and chemokines generated, via the two TLR agonists. TLR2 activation caused slower induction of the NF-kappa B and mitogen-activated protein kinase signaling pathways and yet a much enhanced and prolonged macrophage-inflammatory protein 1 alpha (CC chemokine ligand 3) protein production, when compared with TLR4 stimulation. Enhanced macrophage-inflammatory protein 1 alpha production may contribute to the prolonged down-regulation of CCR1 cell surface expression observed in response to the TLR2 agonist, as preventing chemokine generation with the protein synthesis inhibitor cycloheximide, or CCR1 signaling with the receptor antagonist UCB35625, abolished TLR2- and TLR4-induced CCR1 down-modulation. This result suggests an autocrine pathway, whereby TLR activation can induce chemokine production, which then leads to homologous down-regulation of the cognate receptors. This work provides further insights into the mechanisms that regulate leukocyte recruitment and trafficking during TLR-induced inflammatory responses.     

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.     

Endoplasmic reticulum-associated degradation-induced dissociation of class II invariant chain complexes containing a glycosylation-deficient form of p41

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.     

Homeostatic proliferation of a Qa-1b-restricted T cell: a distinction between the ligands required for positive selection and for proliferation in lymphopenic hosts

Naive T cells proliferate in response to self MHC molecules after transfer into lymphopenic hosts, a process that has been termed homeostatic proliferation (HP). Previous studies have demonstrated that HP is driven by low level signaling induced by interactions with the same MHC molecules responsible for positive selection in the thymus. Little is known about the homeostatic regulation of T cells specific for class Ib molecules, including Qa-1 and H2-M3, though it has been suggested that their capacity to undergo homeostatic expansion may be inherently limited. In this study, we demonstrate that naive 6C5 TCR transgenic T cells with specificity for Qa-1(b) have a capacity similar to conventional T cells to undergo HP after transfer into sublethally irradiated mice. Proliferation was largely dependent on the expression of beta(2)-microglobulin, and experiments with congenic recipients expressing Qa-1(a) instead of Qa-1(b) demonstrated that HP is specifically driven by Qa-1(b) and not through cross-recognition of classical class I molecules. Thus, the same MHC molecule that mediates positive selection of 6C5 T cells is also required for HP. Homeostatic expansion, like positive selection, occurs in the absence of a Qa-1 determinant modifier, the dominant self-peptide bound to Qa-1 molecules. However, experiments with TAP(-/-) recipients demonstrate a clear distinction between the ligand requirements for thymic selection and HP. Positive selection of 6C5 T cells is dependent on TAP function, thus selection is presumably mediated by TAP-dependent peptides. By contrast, HP occurs in TAP(-/-) recipients, providing an example where the ligand requirements for HP are less stringent than for thymic selection.     

Further observations on the ultrastructure of Cystosporogenes operophterae (Canning, 1960) (phylum Microsporidia) parasitic in Operophtera brumata L. (Lepidoptera, Geometridae)

Reinvestigation of Cystosporogenes operophterae [J. Parasitol. 46 (1960) 755] by electron microscopy confirmed that development in host cells takes place in a vacuole with a single membrane at its boundary. Although ribosomes were not clustered on this membrane, it is hypothesised that it originates from host endoplasmic reticulum. The dome-shaped anchoring disc, the morphology of the polaroplast and the separation of the polar tube coils from the ribosome-packed cytoplasm are newly described details of spore structure. The polaroplast consists of an outer region of compact lamellae forming 'arms' surrounding an inner region of widely spaced lamellae. The 'arms' extends back into the region of an elongate nucleus. The genera Cystosporogenes and Endoreticulatus were differentiated by their positions in a previously obtained 16S rDNA phylogeny and on the new ultrastructural data.     

Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends

Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage phiKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.     

Infection, reinfection, and vaccination under suboptimal immune protection: epidemiological perspectives

The SIR (susceptible-infectious-resistant) and SIS (susceptible-infectious-susceptible) frameworks for infectious disease have been extensively studied and successfully applied. They implicitly assume the upper and lower limits of the range of possibilities for host immune response. However, the majority of infections do not fall into either of these extreme categories. We combine two general avenues that straddle this range: temporary immune protection (immunity wanes over time since infection), and partial immune protection (immunity is not fully protective but reduces the risk of reinfection). We present a systematic analysis of the dynamics and equilibrium properties of these models in comparison to SIR and SIS, and analyse the outcome of vaccination programmes. We describe how the waning of immunity shortens inter-epidemic periods, and poses major difficulties to disease eradication. We identify a "reinfection threshold" in transmission when partial immunity is included. Below the reinfection threshold primary infection dominates, levels of infection are low, and vaccination is highly effective (approximately an SIR model). Above the reinfection threshold reinfection dominates, levels of infection are high, and vaccination fails to protect (approximately an SIS situation). This association between high prevalence of infection and vaccine failure emphasizes the problems of controlling recurrent infections in high-burden regions. However, vaccines that induce a better protection than natural infection have the potential to increase the reinfection threshold, and therefore constitute interventions with a surprisingly high capacity to reduce infection where reduction is most needed.     

Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein

We developed a rational approach to identify a site in the vesicular stomatitis virus (VSV) glycoprotein (G) that is exposed on the protein surface and tolerant of foreign epitope insertion. The foreign epitope inserted was the six-amino-acid sequence ELDKWA, a sequence in a neutralizing epitope from human immunodeficiency virus type 1. This sequence was inserted into six sites within the VSV G protein (Indiana serotype). Four sites were selected based on hydrophilicity and high sequence variability identified by sequence comparison with other vesiculovirus G proteins. The site showing the highest variability was fully tolerant of the foreign peptide insertion. G protein containing the insertion at this site folded correctly, was transported normally to the cell surface, had normal membrane fusion activity, and could reconstitute fully infectious VSV. The virus was neutralized by the human 2F5 monoclonal antibody that binds the ELDKWA epitope. Additional studies showed that this site in G protein tolerated insertion of at least 16 amino acids while retaining full infectivity. The three other insertions in somewhat less variable sequences interfered with VSV G folding and transport to the cell surface. Two additional insertions were made in a conserved sequence adjacent to a glycosylation site and near the transmembrane domain. The former blocked G-protein transport, while the latter allowed transport to the cell surface but blocked membrane fusion activity of G protein. Identification of an insertion-tolerant site in VSV G could be important in future vaccine and targeting studies, and the general principle might also be useful in other systems.     

Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5' cap and by 5' and 3' viral sequences

The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).