Immunomodulatory functions of type I interferons

Interferon-α (IFNα) and IFNβ, collectively known as type I IFNs, are the major effector cytokines of the host immune response against viral infections. However, the production of type I IFNs is also induced in response to bacterial ligands of innate immune receptors and/or bacterial infections, indicating a broader physiological role for these cytokines in host defence and homeostasis than was originally assumed. The main focus of this Review is the underappreciated immunomodulatory functions of type I IFNs in health and disease. We discuss their function in the regulation of innate and adaptive immune responses, the response to bacterial ligands, inflammasome activation, intestinal homeostasis and inflammatory and autoimmune diseases.     

Killing the messenger: The role of CXCR7 in regulating primordial germ cell migration

Primordial Germ Cell (PGC) migration in zebrafish is guided by SDF-1a. Binding of this chemokine to its receptor CXCR4b activates downstream signalling cascades leading to cell polarization and directed migration towards the attractant source. Despite the detailed information available concerning the role of SDF-1 in guiding the PGCs to their targets, little was known regarding the molecular mechanisms controlling the distribution of SDF-1a within the tissue. We have recently shown that the activity of a second SDF-1/CXCL12 receptor, CXCR7 is crucial for proper migration of PGCs. Although CXCR4 and CXCR7 are structurally related and serve as receptors for the same ligand, they appear to serve very different functions during PGC migration. Here we discuss a model according to which CXCR4b translates the polarized distribution of SDF-1 into directed PGC migration, while CXCR7 acts as a high-affinity decoy receptor and facilitates the migration of PGCs by shaping the distribution of the chemokine in the environment.Key words: cell migration, CXCR4, CXCR7, SDF-1, chemokine, chemotaxisChemokine-guided cell migration is central for many processes in normal development and homeostasis (e.g., embryogenesis) as well as in pathological conditions (e.g., inflammation). Zebrafish primordial germ cells (PGCs) serve as a useful model for studying chemokine-controlled cell migration in vivo as the migrating PGCs sense and respond to the dynamic distribution of the chemokine SDF-1a through its receptor CXCR4b.^1,2Recent reports identified CXCR7 as a receptor for SDF-1^3,4 that controls processes such as cell adhesion, survival and tumor progression. A role for this receptor in regulating cell migration during development was demonstrated in the zebrafish lateral line.^5,6 The zebrafish lateral line primordium migrates directionally on a stripe of uniform sdf-1a expression to deposit a set of sensory organs along the fish tail. While the authors raised the hypothesis that antagonistic interactions between CXCR4b and CXCR7 polarize the developing organ to allow its migration, the precise function of CXCR7 in this process remained unclear.To address this question in an in vivo context, we examined the role CXCR7 plays in zebrafish PGC migration.⁷ Our experiments revealed that knockdown of cxcr7 translation using morpholino antisense oligo nucleotides results in impaired polarity and aberrant migration of PGCs. Unlike cxcr4b, cxcr7 is not specifically expressed in the PGCs but is initially uniformly distributed throughout the embryo. Furthermore, in contrast to activity of CXCR4, CXCR7 function was found to be required in tissues surrounding the migrating cells rather than in the PGCs themselves.To examine the function of CXCR7 in somatic cells we determined the subcellular localization of the protein as compared with that of CXCR4b and SDF-1a. Interestingly, while CXCR4b is predominantly localized to the plasma membrane, CXCR7 is found primarily in intracellular structures. The fact that SDF-1α and CXCR7 colocalized in the cell and that SDF-1α was found in vesicles that contained the lysosomal marker LAMP-1 suggested that the prime role of CXCR7 is to bind and internalize SDF-1a thereby controlling the level of the diffusible chemokine in the extracellular space. Indeed, observing PGCs expressing CXCR4b on their membrane we detected strong receptor internalization when CXCR7 function was knocked down. The enhanced internalization, a typical response to high levels of SDF-1a⁸ could be reversed by concomitant removal of SDF-1.These findings provided an explanation for the CXCR7 knock-down phenotype as abnormally high levels of SDF-1a in the environment have been shown before to interfere with cell motility.^1,2 Indeed, PGCs in CXCR7 knocked-down embryos displayed strong inhibition of motility manifested in short migration tracks—a phenotype that could be reversed by simultaneous removal of CXCR7 and SDF-1.The implication of the results presented above is that the sole function of CXCR7 in the context of PGC migration is ligand sequestration. Consistent with this idea, two typical signalling responses acting downstream of chemokine receptors namely, elevation of intracellular calcium levels and PI3K activation^9–13 were not altered in cells knocked down for CXCR7. Thus, consistent with other reports,^4,14 our results imply that CXCR7 signalling is not required for PGC migration.An important outstanding question concerns the molecular basis for the dramatic difference between the activity of CXCR4 and that of CXCR7. Defining domains and amino acids responsible for this difference would provide extensive information regarding chemokine receptor signalling and trafficking within the cell. Whereas random mutagenesis and generation of various CXCR4-CXCR7 chimeric molecules might provide an answer to this question, it is tempting to speculate that known protein motifs are responsible for the differences between the two receptors. For example, an obvious candidate region is that around its DRY motif,¹⁴ a motif within the second intracellular loop that is important for Gprotein coupling and signalling.¹⁵ Whereas uncoupling downstream signalling in the case of CXCR7 is an interesting research avenue, other non-mutually exclusive options should be examined (Fig. 1). For example, CXCR7 could possess domains that facilitate interaction with components that enhance internalization. Such an interaction could remove the receptor from the location where it normally interacts with the signalling machinery, while effectively internalizing SDF-1a.Open in a separate windowFigure 1Proposed model for differential functions of CXCR4b and CXCR7. (A) CXCR4b signalling in PGCs controls cell polarization and directional migration in response to SDF-1a binding (squares), through interaction with G-proteins and elevation of calcium levels. (B) Binding of SDF-1a by CXCR7 does not elicit signalling. Endocytosis of the lignad-bound CXCR7 leads to sequestration and degradation of SDF-1a in the somatic environment.Taken together, we show that proper PGC migration requires a mechanism to remove the guidance cue thereby allowing the formation of an informative chemotactic gradient. It would be very interesting to examine whether the paradigm demonstrated for the PGC migration model applies for other chemokine-guided events in development and disease.     

Cell division activity during apical hook development

Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells grow at different rates to form a hook-like structure, which is traditionally assumed to result from differential cell elongation. Using new tools we show asymmetric distribution of cell division during early stages of hook development. Cell divisions in the apical hook were predominantly found in subepidermal layers during an early step of hook development, but were absent in mutants exhibiting a hookless phenotype. In addition, during exaggeration of hook curvature, which is mediated by ethylene, a rapid change in the combined activities of cell division and cell elongation was detected. Our results indicate a fast change in cell division activity during apical hook development. We suggest that cell division together with cell elongation contributes to apical hook growth. Our results emphasize the change in the relative contribution of cell division and cell elongation in a fast growing structure like the apical hook.     

Regulation of fibroblast cyclooxygenase synthesis by interleukin-1

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.     

Imidazole: a selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 MICRONG/ML; DETERMINED BY BIOASSAY). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PGH2. Several imidazole analogues and other substances were tested but only 1-methyl-imidazole was more potent than imidazole itself. The use of imidazole to inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

Prostaglandin biosynthesis in rabbit kidney medulla: Inhibition vs. by aspirin, indomethacin and meclofenamic acid

The non-steroidal anti-inflammatory drugs aspirin, indomethacin and meclofenamic acid were compared for their potency and duration of inhibition of prostaglandin biosynthesis in rabbit kidney medulla. Indomethacin and meclofenamic acid showed equal potency of inhibition (IC₅₀ 0.88 μM and 0.85 μM respectively) while aspirin was a much weaker inhibitor (IC₅₀ 120 μM). , indomethacin was the most powerful inhibitor (ID₅₀ 0.034 mg/kg) followed by meclofenamic acid (0.45 mg/kg) and aspirin (2.35 mg/kg).Studies on the duration of inhibition by these compounds showed the effect of indomethacin and meclofenamic acid to be completely reversed within 4–6 hours. In contrast, return of kidney prostaglandin biosynthetic activity following aspirin inhibition is very slow and significant inhibition is still present 48 hours after a single aspirin injection. The inhibitory effect of aspirin could be blocked by pretreatment with indomethacin, indicating that both drugs interact with related sites on the cyclo-oxygenase enzyme. The irreversible inhibition of the cyclo-oxygenase by aspirin as demonstrated in studies of other investigators suggests that the return of kidney prostaglandin synthetase activity after aspirin inhibition represents synthesis of new cyclo-oxygenase protein.     

Prostaglandin biosynthesis and lipolysis in subcellular fractions from rabbit kidney medulla.

Three separate prostaglandin-generating activities are associated with plasma membranes, mitochondria and microsomal fractions from rabbit kidney medulla. In the plasma membranes and mitochondria, but not in microsomal fractions, Ca2+ ions stimulate the activity of phospholipase A2, yielding selective release of arachidonic acid and linoleic acid and concomitant increase in prostaglandin E2 formation.