William Paul

Defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain

The common gamma chain (gamma c) of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors is defective in humans with XSCID. Mice lacking gamma c expression had hypoplastic thymuses; the thymocytes responded to gamma c-independent mitogens, but not gamma c-dependent stimuli. Splenic T cells were diminished at 3 weeks of age, but CD4+ T cells markedly increased by 4 weeks. B cells were greatly diminished in contrast with the situation in XSCID. NK cells, gamma delta intestinal intraepithelial lymphocytes, dendritic epidermal T cells, peripheral lymph nodes, and gut-associated lymphoid tissue were absent. These findings underscore the importance of gamma c in lymphoid development. Moreover, differences in humans and mice lacking gamma c expression indicate species-specific differences in the roles of gamma c-dependent cytokines or in the existence of redundant pathways. These mice provide an important model for studying the pathophysiology provide an important model for studying the pathophysiology of and gene therapy for human XSCID.

Inflammatory actions of platelet activating factor (Paf-acether) in guinea-pig skin

1 Cutaneous responses to synthetic platelet activating factor (Paf‐acether) have been studied in guinea‐pigs by means of radioisotopic marker techniques. 2 Intradermal injection of Paf‐acether elicited increased plasma protein extravasation (IPPE) (0.2–200 pmol/site), platelet accumulation (PA) (20–200 pmol/site) and red blood cell accumulation (RBCA) (200 pmol/site), whereas lyso‐Paf (up to 2 nmol/site) was inactive in all these respects. 3 Following intradermal injection, the IPPE responses to Paf‐acether (2 and 20 pmol/site) were complete within 15 and 30 min respectively, although in response to 200 pmol/site, IPPE was detectable up to 1.5 h. The PA and RBCA responses to Paf‐acether (200 pmol/site) were complete within 1 h. 4 IPPE induced by Paf‐acether (3 pmol/site) was potentiated by concomitant intradermal injection of a cutaneous vasodilator prostaglandin E2 (PGE2, 1 nmol/site) and inhibited by the β‐adrenoceptor agonist, isoprenaline (4.5 nmol/site) or the α‐adrenoceptor agonist, phenylephrine (6 nmol/site). Such observations are consistent with Paf‐acether effecting increased vessel wall permeability. 5 Intradermal injection of PGEx (3 nmol/site) significantly reduced PA in response to Paf‐acether (200 pmol/site), whilst significantly enhancing IPPE. This dissociation of increased vascular permeability from PA is consistent with Paf‐acether eliciting IPPE via a platelet‐independent mechanism. 6 These results indicate that a direct effect on vessel wall permeability contributes to the inflammatory response to Paf‐acether in guinea‐pig skin. It is suggested that Paf‐acether is a potential mediator of allergy and inflammation.

Platelet‐activating factor: a possible mediator of the dual response to allergen?

Certain allergic asthmatic patients exhibit a dual response in the lung following bronchial challenge with the appropriate allergen, Often this is paralleled by a cutaneous dual response when the antigen is injected intradermally. The mechanisms underlying such phenomena are not established, but some evidence suggests that the late response is a consequence of the early response. Since platelet activation has been observed following antigen challenge in asthmatic subjects, we have studied the ability of platelet activating factor (PAF‐acether. AGEPC) to induce cutaneous inflammatory responses in man. In a time course study over 24 hr, PAF‐acether produced a biphasic response: an immediate weal and flare reaction, which resolved within 1‐2 hr and was followed some 3‐6 hr later by a delayed reaction in which erythema associated with hyperaigesia was evident. These observations suggest that PAF‐acether should be considered in the context of allergic asthma as a possible mediator of the dual response to allergen.

Formation of IL-7Ralphahigh and IL-7Ralphalow CD8 T cells during infection is regulated by the opposing functions of GABPalpha and Gfi-1

IL-7 is essential for the survival of naive and memory T cells, and IL-7 receptor α-chain (IL-7Rα) expression is dynamically regulated in activated CD8 T cells during acute viral and bacterial infections. Most virus-specific CD8 T cells become IL-7Rαlow and are relatively short-lived, but some escape IL-7Rα repression (referred to as IL-7Rαhigh memory precursor effector cells) and preferentially enter the memory CD8 T cell pool. How antiviral effector CD8 T cells regulate IL-7Rα expression in an “on and off” fashion remains to be characterized. During lymphocytic choriomeningitis virus infection, we found that opposing actions of the transcription factors GABPα (GA binding protein α) and Gfi-1 (growth factor independence 1) control IL-7Rα expression in effector CD8 T cells. Specifically, GABPα was required for IL-7Rα expression in memory precursor effector cells, and this correlated with hyperacetylation of the Il7ra promoter. In contrast, Gfi-1 was required for stable IL-7Rα repression in effector CD8 T cells and acted by antagonizing GABPα binding and recruiting histone deacetylase 1, which deacetylated the Il7ra promoter. Thus, Il7ra promoter acetylation and activity was dependent on the reciprocal binding of GABPα and Gfi-1, and these data provide a biochemical mechanism for the generation of stable IL-7Rαhigh and IL-7Rαlow states in virus-specific effector CD8 T cells.

Prevention of pulmonary thromboembolism by NCX 4016, a nitric oxide-releasing aspirin.

We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.

Cutting edge: lack of high affinity competition for peptide in polyclonal CD4+ responses unmasks IL-4 production.

Priming of naive monoclonal CD4 T cells via weak agonsim permits GATA-3 transcription and Th2 differentiation. To test whether this process can occur in polyclonal naive populations, where a range of TCR affinities exists for any given Ag/MHC complex, we primed naive CD4 cells from 5CC7 Vβ3 transgenic mice, which have a fixed β-chain specific for pigeon cytochrome c peptide I-Ek. Priming populations de-pleted of higher affinity, moth cytochrome c pep-tide I-Ek tetramer-binding cells resulted in substantial IL-4 production that did not occur in the presence of higher affinity cells. TCRα-chain sequence analysis showed that clones that possessed TCR features associated with high affinity responses to pigeon cytochrome c made less IL-4 than clones that possessed fewer such motifs. These results indicate that cells bearing TCRs that are weakly stimulated by their cognate Ag preferentially adopt a Th2 phenotype when primed in the absence of competition from cells with higher affinity receptors.

Individual T Helper Cells Have a Quantitative Cytokine Memory

The probabilistic expression of cytokine genes in differentiated T helper (Th) cell populations remains ill defined. By single-cell analyses and mathematical modeling, we show that one stimulation featured stable cytokine nonproducers as well as stable producers with wide cell-to-cell variability in the magnitude of expression. Focusing on interferon-γ (IFN-γ) expression by Th1 cells, mathematical modeling predicted that this behavior reflected different cell-intrinsic capacities and not mere gene-expression noise. In vivo, Th1 cells sort purified by secreted IFN-γ amounts preserved a quantitative memory for both probability and magnitude of IFN-γ re-expression for at least 1 month. Mechanistically, this memory resulted from quantitatively distinct transcription of individual alleles and was controlled by stable expression differences of the Th1 cell lineage-specifying transcription factor T-bet. Functionally, Th1 cells with graded IFN-γ production competence differentially activated Salmonella-infected macrophages for bacterial killing. Thus, individual Th cells commit to produce distinct amounts of a given cytokine, thereby generating functional intrapopulation heterogeneity.

Increased platelet aggregation in vivo in the Zucker Diabetic Fatty rat: differences from the streptozotocin diabetic rat

Diabetes mellitus, especially type 2, is associated with increased arterial thrombosis. Our aims were (i) to characterize and compare platelet aggregation in vivo and in vitro in a type 2 diabetes model; and (ii) to determine whether these results differ from those in a type 1 diabetes model.

Regulation of platelet function by catecholamines in the cerebral vasculature of the rabbit

111In‐labelled platelets were monitored continuously in the cerebral and pulmonary vascular beds of anaesthetized rabbits. Dopamine can, depending upon the concentration, either potentiate or inhibit thrombin‐induced platelet accumulation in the cerebral vasculature of rabbits by unknown mechanisms. The effects of specific adrenergic and dopaminergic receptor antagonists were tested upon dopamines actions on intracarotid (i.c.) thrombin‐induced (80u2003uu2003kg−1) platelet accumulation in the cerebral vasculature. The effect of adrenaline on the response to thrombin in this vascular bed was also investigated. Thrombin‐induced platelet accumulation was significantly (P<0.01) potentiated by dopamine (100u2003μgu2003kg−1u2003min−1, i.c.) and this effect was significantly inhibited by infusion of the α‐adrenoceptor antagonist, phentolamine. A higher dose of dopamine (2u2003mgu2003kg−1u2003min−1, i.c.) inhibited thrombin‐induced platelet accumulation. The β‐adrenoceptor antagonist, propranolol, did not significantly alter this inhibitory effect whereas it was abolished by the dopamine D1 selective antagonist, SCH23390. Adrenaline (when administered i.c. by bolus injection or infusion) had no significant effect on thrombin‐induced accumulation at any of the doses tested. Potentiation of in vivo platelet accumulation by dopamine therefore seems to occur via α‐adrenergic receptors. However, the inhibitory effect of dopamine appears to be exerted via the activation of dopamine D1 receptors and not via β‐adrenergic receptors. Our findings confirm that dopamine, but not adrenaline, can modify platelet function in the cerebral vasculature and these observations may have implications for current and potential therapeutic uses of dopamine and selective dopaminergic compounds.

The effects of heparin and related molecules on vascular permeability and neutrophil accumulation in rabbit skin

Unfractionated heparin (UH) has been shown to possess a wide range of properties which are potentially anti‐inflammatory. Many of these studies, including effects of heparin on adhesion of inflammatory cells to endothelium, have been carried out in vitro. In the present study, we have used radioisotopic techniques to study the effect of UH, and related molecules, on in vivo inflammatory responses (plasma exudation (PE) and PMN accumulation) in rabbit skin induced by cationic proteins, mediators and antigen. Intradermal (i.d.) pretreatment with UH dose‐dependently inhibited poly‐L‐lysine (PLL)‐induced responses. The same treatment had no effect on antigen (extract of Alternaria tenuis, AT)‐, formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐ or leukotriene (LT) B4‐induced responses, although i.d. dextran sulphate (DS) significantly inhibited responses to all of these mediators. High dose (10,000u2003uu2003kg−1) intravenous UH significantly decreased cutaneous responses to fMLP and LTB4. By comparison, the selectin inhibitor, fucoidin, and DS, were very effective inhibitors of these responses, and of responses to AT and PLL. In contrast to the weak effect in the in vivo studies, UH significantly inhibited in vitro homotypic aggregation of rabbit PMNs, showing that it can modify PMN function. Our data with i.d. UH confirm the important ability of this molecule to interact with and neutralize polycationic peptides in vivo, suggesting that this is a prime role of endogenous heparin. The lack of effect of exogenous heparin on acute inflammatory responses induced by allergen, suggests that cationic proteins are unlikely to be primary mediators of the allergen‐induced PE or PMN accumulation.