Intrarectal immunization and IgA antibody-secreting cell homing to the small intestine.

According to the current paradigm, lymphocyte homing to the small intestine requires the expression of two tissue-specific homing receptors, the integrin α4ß7 and the CCL25 receptor CCR9. In this study, we investigated the organ distribution and the homing molecule expression of IgA Ab-secreting cells (ASCs) induced by intrarectal immunization with a particulate Ag, in comparison with other mucosal immunization routes. Intrarectal immunization induces gut-homing IgA ASCs that localize not only in the colon but also in the small intestine, although they are not responsive to CCL25, unlike IgA ASCs induced by oral immunization. The mucosal epithelial chemokine CCL28, known to attract all IgA ASCs, does not compensate for the lack of CCL25 responsiveness, because the number of Ag-specific cells is not decreased in the gut of CCR10-deficient mice immunized by the intrarectal route. However, Ag-specific IgA ASCs induced by intrarectal immunization express the integrin α4ß7, and their number is considerably decreased in the gut of ß7-deficient mice immunized by the intrarectal route, indicating that α4ß7 enables these cells to migrate into the small intestine, even without CCL25 responsiveness. In contrast, IgA ASCs induced by intranasal immunization express low α4ß7 levels and are usually excluded from the gut. Paradoxically, after intranasal immunization, Ag-specific IgA ASCs are significantly increased in the small intestine of ß7-deficient mice, demonstrating that lymphocyte homing is a competitive process and that integrin α4ß7 determines not only the intestinal tropism of IgA ASCs elicited in GALTs but also the intestinal exclusion of lymphocytes primed in other inductive sites.

Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants.

Murine norovirus (MNV) was used as a surrogate to study resistance of human norovirus to disinfectants used in hospitals. MNV was sensitive to alcohol, alcohol hand rubs, bleach, and povidone iodine-based disinfectant. Real-time reverse transcription-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious virus.

Cytohesin binder and regulator (cybr) is not essential for T- and dendritic-cell activation and differentiation.

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

Intrarectal immunization with rotavirus 2/6 virus-like particles induces an antirotavirus immune response localized in the intestinal mucosa and protects against rotavirus infection in mice.

Rotavirus (RV) is the main etiological agent of severe gastroenteritis in infants, and vaccination seems the most effective way to control the disease. Recombinant rotavirus-like particles composed of the viral protein 6 (VP6) and VP2 (2/6-VLPs) have been reported to induce protective immunity in mice when administered by the intranasal (i.n.) route. In this study, we show that administration of 2/6-VLPs by the intrarectal (i.r.) route together with either cholera toxin (CT) or a CpG-containing oligodeoxynucleotide as the adjuvant protects adult mice against RV infection. Moreover, when CT is used, RV shedding in animals immunized by the i.r. route is even reduced in comparison with that in animals immunized by the i.n. route. Humoral and cellular immune responses induced by these immunization protocols were analyzed. We found that although i.r. immunization with 2/6-VLPs induces lower RV-specific immunoglobulin G (IgG) and IgA levels in serum, intestinal anti-RV IgA production is higher in mice immunized by the i.r. route. Cellular immune response has been evaluated by measuring cytokine production by spleen and Peyer's patch cells (PPs) after ex vivo restimulation with RV. Mice immunized by the i.n. and i.r. routes display higher gamma interferon production in spleen and PPs, respectively. In conclusion, we demonstrate that i.r. immunization with 2/6-VLPs protects against RV infection in mice and is more efficient than i.n. immunization in inducing an anti-RV immune response in intestinal mucosa.

Jak3-independent trafficking of the common gamma chain receptor subunit: chaperone function of Jaks revisited.

Janus kinases (Jaks) play an essential role in cytokine signaling and have been reported to regulate plasma membrane expression of their cognate receptors. In this study, we examined whether Jak3 and the common gamma chain (gamma(c)) reciprocally regulate their plasma membrane expression. In contrast to interleukin-2Ralpha, gamma(c) localized poorly to the plasma membrane and accumulated in endosomal-lysosomal compartments. However, gamma(c) was expressed at comparable levels on the surface of cells lacking Jak3, and plasma membrane turnover of gamma(c) was independent of Jak3. Nonetheless, overexpression of Jak3 enhanced accumulation of gamma(c) at the plasma membrane. Without gamma(c), Jak3 localized in the cytosol, whereas in the presence of the receptor, it colocalized with gamma(c) in endosomes and at the plasma membrane. Although the Jak FERM domain is necessary and sufficient for receptor binding, the requirement for full-length Jak3 in gamma(c) membrane trafficking was remarkably stringent; using truncation and deletion mutants, we showed that the entire Jak3 molecule was required, although kinase activity was not. Thus, unlike other cytokine receptors, gamma(c) does not require Jak3 for receptor membrane expression. However, full-length Jak3 is required for normal trafficking of this cytokine receptor/Jak pair, a finding that has important structural and clinical implications.

Granulocyte colony-stimulating factor decreases tumor necrosis factor production in whole blood: role of interleukin-10 and prostaglandin E(2).

Previous reports have indicated that the administration of granulocyte colony-stimulating factor (G-CSF) decreases ex vivo tumor necrosis factor (TNF) production in humans. In this study, we report that daily pretreatment of mice with G-CSF for three days decreases ex vivo lipopolysaccharide (LPS)-induced TNF production in whole blood. Conversely, production of interleukin-10 (IL-10) and prostaglandin E(2) (PGE(2)) is increased. The inhibitory effect of G-CSF pretreatment on TNF production is partially reversed by addition of an anti-IL-10 antibody, and completely reversed by combined addition of anti-IL-10 antibody and the cyclooxygenase (COX) inhibitor, ketoprofen. These results suggest that G-CSF decreases TNF production in this experimental model by increasing production of IL-10 and PGE(2), which are both known inhibitors of TNF production.

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.

Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights.

The differentiation of naive CD4+ T cells into subsets of T helper cells is a pivotal process with major implications for host defense and the pathogenesis of immune-mediated diseases. Though the basic paradigm was discovered more than 15 years ago, new discoveries continue to be made that offer fresh insights into the regulation of this process. T helper (TH)1 cells produce interferon (IFN)-gamma, promoting cell-mediated immunity and control of intracellular pathogens. We now know that TH1 differentiation is regulated by transcription factors such as T-bet, Stat1, and Stat4, as well as cytokines such as IL-12, IL-23, IL-27, type I IFNs, and IFN-gamma. In contrast, TH2 cells produce IL-4, which promotes allergic responses and is important in host defense against helminths. The transcription factors Stat6, GATA-3, c-Maf, NFATs, and the cytokine IL-4 promote TH2 differentiation. These key regulators of TH differentiation are the subject of this review.

Erythropoietin exerts an anti-inflammatory effect on the CNS in a model of experimental autoimmune encephalomyelitis.

In recent work we reported that systemically administered erythropoietin (EPO) crosses the blood-brain barrier and has protective effects in animal models of cerebral ischemia, brain trauma and in a rat model of experimental autoimmune encephalomyelitis (EAE). Here we characterize the effect of systemic EPO on the inflammatory component of actively induced, acute EAE in Lewis rats. Administration of EPO at doses of 500-5000 U/kg bw i.p., daily from day 3 after immunization with myelin basic protein (MBP), delayed the onset of EAE and decreased its clinical score at peak time (days 12-13). Immunohistochemical analysis of the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-CD11b antibodies showed that EPO markedly diminished inflammation and glial activation/proliferation. EAE induced significant levels of TNF and IL-6 in the spinal cord, where IL-6 was maximum at the onset of the disease (day 10) and TNF at its peak (day 12). EPO delayed the increase of TNF levels, without altering their peak levels, and markedly reduced those of IL-6 suggesting that the decreased inflammation and clinical score may be in part upon attenuation of IL-6. On the other hand, EPO was without effect in a model of adjuvant-induced arthritis in Lewis rats, suggesting a specificity towards autoimmune demyelinating diseases. These data suggest that EPO might act as a protective cytokine in inflammatory pathologies of the CNS.

HMGB-1, a DNA-binding protein with cytokine activity, induces brain TNF and IL-6 production, and mediates anorexia and taste aversion.

High-mobility group protein-1 (HMG-1 also termed HMGB-1), a DNA-binding protein, regulates gene transcription and stabilizes nucleosome formation. HMG-1 was recently implicated as a cytokine, because it is a late-acting mediator of endotoxin lethality that induces the release of pro-inflammatory cytokines from monocytes. Here it is shown that administration of HMG-1 into the cerebral ventricles decreases food intake (food intake=4.6g/mouse in controls vs 1.6g/mouse after 1 microg HMG-1 i.c.v.; P <0.05). Intracerebroventricular HMG-1 induced an increased in TNF and IL-6 expression in the brain, and mediated taste aversion with potencies equivalent to LPS. In a model of endotoxemia, passive immunization with anti-HMG-1 antibodies attenuated the development of hypophagia, indicating that HMG-1 is a mediator of sickness behaviour associated with endotoxemia.

Preventive administration of Mycobacterium tuberculosis 10-kDa heat shock protein (hsp10) suppresses adjuvant arthritis in Lewis rats.

Adjuvant arthritis (AA) can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt) in oil. We have investigated the modulation of AA by mycobacterial 10-kDa heat shock protein (hsp10), administered according to several protocols known to induce immune tolerance and immune deviation. Subcutaneous immunization with hsp10 in aqueous solution did not induce a cellular immune response, evaluated as delayed-type hypersensitivity (DTH) reaction, although anti-hsp10 antibodies, mainly of the IgG2a isotype, were detected in serum of treated animals. When rats were pretreated with hsp10 in aqueous solution before AA induction, no effects were seen on arthritis-induced joint swelling, although osteolysis and lymphocyte infiltration were slightly decreased. When other routes of administration were attempted, the strongest suppression was seen in the group of animals which received four intranasal (i.n.) administrations of protein and a subsequent challenge of hsp10 in incomplete Freund's adjuvant (IFA). We also found that the extent of disease suppression among the different groups of animals correlated with serum anti-hsp10 antibody levels. These antibodies mostly belonged to the IgG2a subtype, suggesting that immune deviation may play a role in the mechanism of disease suppression by hsp10.

Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity.

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5prime prime or minute-O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).