Biomedical subjects

Raif S Geha

Publications and source records attributed to Raif S Geha.

At least 19 recordsLinked to original sources

PubMed2007

WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP).

Wiskott-Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP(-/-) mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP(-/-) mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP(-/-) mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

Actins↗

PubMed2006

Interleukin receptor-associated kinase-4 deficiency impairs Toll-like receptor-dependent innate antiviral immune responses.

BACKGROUND: Engagement of all known Toll-like receptors (TLRs) causes the production of inflammatory cytokines, including TNF-alpha, whereas in humans, engagement of TLRs 3, 7, 8, and 9 also induces type I IFNs. IRAK-4 is a critical effector in signaling by TLRs and the IL-1 receptor, which share homology in their intracellular domain and recruit IRAK-4 via the adaptor myeloid differentiation factor 88 (MyD88). Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections but have so far not been reported to be susceptible to viral infection. Blood cells from these patients are impaired in their ability to make TNF-alpha in response to activation by TLRs. A recent report has described concomitant impairment of type I IFN production after activation of TLRs 7, 8, and 9, but not TLR3. OBJECTIVES: We sought to evaluate the role of IRAK-4 in TLR-induced production of the type I IFN, IFN-alpha, in humans. METHODS: We examined TLR-induced production of TNF-alpha and IFN-alpha in PBMCs from an IRAK-4-deficient patient, his heterozygous carrier parents, and normal controls. RESULTS: TNF-alpha production in response to TLR agonists was severely impaired in the patient. IFN-alpha production induced by TLR7, TLR8, and TLR9, as well as TLR3 agonists, was low or absent. CONCLUSIONS: IRAK-4 plays an important role in the production of type I IFN, as well as TNF-alpha, induced by all TLRs, including TLR3. CLINICAL IMPLICATIONS: IRAK-4 may play a broader role in human innate antiviral immunity than previously appreciated.

Cells, Cultured↗

PubMed2006

CD1d restricted natural killer T cells are not required for allergic skin inflammation.

BACKGROUND: Invariant T-cell receptor-positive natural killer (iNKT) cells have been shown to be essential for the development of allergen-induced airway hyperreactivity (AHR). OBJECTIVE: We examined the role of iNKT cells in allergic skin inflammation using a murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). METHODS: Wild-type (WT) and natural killer T-cell-deficient CD1d-/- mice were epicutaneously sensitized with OVA or normal saline and challenged with aerosolized OVA. iNKT cells in skin and bronchoalveolar lavage fluid were analyzed by fluorescence-activated cell sorting, and cytokine mRNA levels were measured by quantitative RT-PCR. AHR to methacholine was measured after OVA inhalation. RESULTS: Skin infiltration by eosinophils and CD4+ cells and expression of mRNA encoding IL-4 and IL-13 in OVA-sensitized skin were similar in WT and CD1d-/- mice. No significant increase in iNKT cells was detectable in epicutaneously sensitized skin. In contrast, iNKT cells were found in the bronchoalveolar lavage fluid from OVA-challenged epicutaneously sensitized WT mice, but not CD1d-/- mice. Epicutaneously sensitized CD1d-/- mice had an impaired expression of IL-4, IL-5, and IL-13 mRNA in the lung and failed to develop AHR in response to airway challenge with OVA. CONCLUSION: These results demonstrate that iNKT cells are not required for allergic skin inflammation in a murine model of AD, in contrast with airway inflammation, in which iNKT cells are essential. CLINICAL IMPLICATIONS: Understanding the potential role of iNKT cells in AD will allow us to have a more specific target for therapeutic use.

Allergens↗

PubMed2006

The H1 histamine receptor regulates allergic lung responses.

Histamine, signaling via the type 1 receptor (H1R), has been shown to suppress Th2 cytokine production by in vitro cultured T cells. We examined the role of H1R in allergic inflammation in vivo using a murine asthma model. Allergen-stimulated splenic T cells from sensitized H1R-/- mice exhibited enhanced Th2 cytokine production. Despite this Th2 bias, allergen-challenged H1R-/- mice exhibited diminished lung Th2 cytokine mRNA levels, airway inflammation, goblet cell metaplasia, and airway hyperresponsiveness (AHR). Restoration of pulmonary Th2 cytokines in H1R-/- mice by intranasal IL-4 or IL-13 restored inflammatory lung responses and AHR. Further investigation revealed that histamine acts as a T cell chemotactic factor and defective T cell trafficking was responsible for the absence of lung inflammation. Cultured T cells migrated in response to histamine in vitro, but this was ablated by blockade of H1R but not H2R. In vivo, allergen-specific WT but not H1R-/- CD4+ T cells were recruited to the lungs of naive recipients following inhaled allergen challenge. H1R-/- T cells failed to confer airway inflammation or AHR observed after transfer of WT T cells. Our data establish a role for histamine and H1R in promoting the migration of Th2 cells into sites of allergen exposure.

Administration, Intranasal↗

PubMed2006

The complement component C3 plays a critical role in both Th1 and Th2 responses to antigen.

BACKGROUND: Complement component C3 is synthesized by keratinocytes and is activated after skin injury. C3 is also synthesized by peritoneal macrophages, which are activated by the adjuvant alum. OBJECTIVE: We sought to investigate the role of C3 in inciting allergic skin Inflammation and systemic immune responses after epicutaneous sensitization or intraperitoneal sensitization with antigen. METHODS: C3-deficient (C3-/-) mice and wild-type (WT) control animals were subjected to epicutaneous sensitization with the antigen ovalbumin (OVA) on shaved and tape-stripped skin or intraperitoneal immunization with OVA in alum. RESULTS: Skin Infiltration by eosinophils and expression of mRNA encoding the TH2 cytokines IL-4 and IL-5 in OVA-sensitized skin sites was impaired in C3-/- mice. Splenocytes from epicutaneously sensitized C3-/- mice secreted less IL-4, IL-5, IL-13, and IFN-gamma in response to OVA stimulation than splenocytes from WT control animals. The defect in cytokine secretion by splenocytes was also observed after intraperitoneal immunization of C3-/- mice. C3-/- mice had impaired IgG1, IgG2a, and IgE antibody responses after both epicutaneous and intraperitoneal immunization. The defect in cytokine secretion of C3-/- mice was not due to defective proliferation to antigen, was not observed after anti-CD3 stimulation, and was corrected by the addition of purified C3 protein. CONCLUSION: These results suggest that C3 plays an important role in both the TH1 and TH2 response to antigen in vivo. CLINICAL IMPLICATIONS: The complement pathway might be a potential target in the therapy of allergic diseases.

Administration, Cutaneous↗

PubMed2006

A second-site mutation in the initiation codon of WAS (WASP) results in expansion of subsets of lymphocytes in an Wiskott-Aldrich syndrome patient.

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the gene encoding WAS protein (WASP ). Recently, somatic mosaicism caused by reversions or second-site mutations has been reported in some inherited disorders including WAS. In this article, we describe somatic mosaicism in a 15-year-old WAS patient due to a second-hit mutation in the initiation codon. The patient originally had a single-base deletion (c.11delG; p.G4fsX40) in the WAS (WASP) gene, which resulted in a frameshift and abrogated protein expression. Subsequently, a fraction of T and natural killer (NK) cells expressed a smaller WASP, which binds to its cellular partner WASP-interacting protein (WIP). The T and NK cells were found to have an additional mutation in the initiation codon (c.1A>T; p.M1_P5del). The results strongly suggest that the smaller WASP is translated from the second ATG downstream of the original mutation, and not only T cells but also NK cells carrying the second mutation acquired a growth advantage over WASP negative counterparts. To our knowledge, this is the first report describing somatic mosaicism due to a second-site mutation in the initiation codon of any inherited disorders.

Adolescent↗

PubMed2006

TACI mutation in common variable immunodeficiency and IgA deficiency.

Common variable immunodeficiency (CVID) is a heterogeneous primary immunodeficiency disease. Immunoglobulin A deficiency (IGAD) shares some clinical, laboratory, and genetic features with CVID and occurs with relatively greater frequency in first-degree relatives of individuals with CVID. Recently, patients with CVID and IGAD have been found to have mutations of the gene TNFRSF13B encoding the TACI (transmembrane activator and calcium-modulator and cyclophilin-ligand interactor), a member of the tumor necrosis factor-receptor superfamily. In this article, we review the various TACI mutations that have been identified so far. Although six mutations have been reported, no clear genotype-phenotype association has been shown to date. This suggests that the phenotypic expression of TACI mutation is affected by additional genetic and environmental factors. Analysis of a larger sample of patients will be needed to determine if the specific mutations are associated with a particular phenotype or predisposition to the common features of CVID and IGAD: autoimmunity, lymphoproliferation, or malignancy.

Animals↗

PubMed2006

2. Update on primary immunodeficiency diseases.

The pace of discovery in primary immunodeficiency continues to accelerate. In particular, lymphocyte defects have been the source of the most impressive expansion in recent years. Novel forms of agammaglobulinemia, class-switch defects, and T-B(+) severe combined immunodeficiency have been described. Little by little, the genetic heterogeneity of the common variable immunodeficiency and IgA deficiency phenotypes continues to be unraveled as new molecular defects have been reported in these patients as well. The phenotypic spectrum of DiGeorge syndrome has been further developed, along with promising advances in therapy. Defects of nuclear factor kappaB regulation and Toll-like receptor signaling have been described, along with defects of chemokine receptors and cytoplasmic proteases. Clinically defined immunodeficiencies, such as hyper-IgE syndrome and idiopathic CD4 lymphocytopenia, are also discussed. Finally, significant adverse effects in some patients have tempered initial enthusiasm for gene therapy.

Agammaglobulinemia↗

PubMed2006

Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005.

Although relatively rare, primary immunodeficiency diseases (PIDs) provide an excellent window into the functioning of the immune system. In the late 1960s, observations on these diseases, with their associated infections and genetics, bisected the immune system into humoral immunity and cell-mediated immunity. These diseases also represent a challenge in their diagnosis and treatment. Beginning in 1970, a unified nomenclature for the then-known PIDs was created by a committee convoked by the World Health Organization. Since then, and later under the aegis of the International Union of Immunological Societies, an international committee of experts has met every 2 to 3 years to update the classification of PIDs. During the past 15 years, the molecular basis of more than 120 PIDs has been elucidated. This update results from the latest meeting of this committee in Budapest, Hungary, in June 2005, which followed 2 1/2 days of scientific discussions. As a result of this work, new entities have been included, and the nomenclature of some PIDs (specifically of the various forms of class-switch recombination defects, previously known as hyper-IgM syndromes) has been changed.

B-Lymphocytes↗

PubMed2006

Molecular basis of common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most prevalent human primary immunodeficiency requiring medical attention. Until recently, the only known genetic defect specific to CVID was the inducible costimulatory receptor (ICOS) deficiency, which accounts for less than 1% of the patients. Recently, mutations in the TNF receptor family member transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), which mediates isotype switching in B cells, were found to be present in 10% to 20% of patients with CVID. Mutations in TACI were also found in relatives of patients with CVID who had IgA deficiency (IgAD), as well as in a patient with isolated IgAD. In the majority of patients described to date, only one TACI allele is mutated, showing an autosomal dominant transmission of the disease. B cells from individuals with TACI mutations did not produce IgG and IgA in response to the TACI ligand a proliferation-inducing ligand (APRIL), probably reflecting impaired isotype switching. These results suggest that TACI mutations can lead to CVID and IgAD.

Animals↗

PubMed2006

3BP2 deficiency impairs the response of B cells, but not T cells, to antigen receptor ligation.

The adapter protein 3BP2 is expressed in lymphocytes; binds to Syk/ZAP-70, Vav, and phospholipase C-gamma (PLC-gamma); and is thought to be important for interleukin-2 gene transcription in T cells. To define the role of 3BP2 in lymphocyte development and function, we generated 3BP2-deficient mice. T-cell development, proliferation, cytokine secretion, and signaling in response to T-cell receptor (TCR) ligation were all normal in 3BP2(-/-) mice. 3BP2(-/-) mice had increased accumulation of pre-B cells in the bone marrow and a block in the progression of transitional B cells in the spleen from the T1 to the T2 stage, but normal numbers of mature B cells. B-cell proliferation, cell cycle progression, PLC-gamma2 phosphorylation, calcium mobilization, NF-ATp dephosphorylation, and Erk and Jnk activation in response to B-cell receptor (BCR) ligation were all impaired. These results suggest that 3BP2 is important for BCR, but not for TCR signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2006

IPEX and the role of Foxp3 in the development and function of human Tregs.

Genetic defects in the transcription factor forkhead box protein P3 (Foxp3) cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). IPEX is thought to be due to a defect in naturally arising CD4+ Tregs. In this issue of the JCI, Bacchetta and colleagues demonstrate that patients with IPEX and missense mutations in Foxp3 provide insight into the role of various domains of Foxp3 in the development and function of Tregs (see the related article beginning on page 1713).

Animals↗

PubMed2005

A 10-aa-long sequence in SLP-76 upstream of the Gads binding site is essential for T cell development and function.

The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.

Adaptor Proteins, Signal Transducing↗

PubMed2005

WIP and WASP play complementary roles in T cell homing and chemotaxis to SDF-1alpha.

Homing of lymphocytes to tissues is a biologically important multistep process that involves selectin-dependent rolling, integrin-dependent adhesion and chemokine-directed chemotaxis. The actin cytoskeleton plays a central role in lymphocyte adhesion and motility. Wiskott-Aldrich syndrome protein (WASP), the product of the gene mutated in Wiskott-Aldrich syndrome, and its partner, the Wiskott-Aldrich syndrome protein-interacting protein (WIP), play important roles in actin re-organization in T lymphocytes. We used mice with disruption of the WASP and WIP genes to examine the role of WASP and WIP in T cell homing. T cell homing to spleen and lymph nodes in vivo was deficient in WASP-/- and WIP-/- mice and severely impaired in WASP-/-WIP-/- double knockout (DKO) mice. Deficiency of WASP, WIP or both did not interfere with selectin-dependent rolling or integrin-dependent adhesion of T cells in vitro. Chemotaxis to stromal cell-derived factor-1alpha (SDF-1alpha) in vitro was mildly reduced in T cells from WASP-/- mice. In contrast, it was significantly impaired in T cells from WIP-/- mice and severely reduced in T cells from DKO mice. Cellular F-actin increase following SDF-1alpha stimulation was normal in WASP-/- and WIP-/- T cells, but severely reduced in T cells from DKO mice. Actin re-organization and polarization in response to SDF-1alpha was abnormal in T cells from all knockout mice. Early biochemical events following SDF-1alpha stimulation that are important for chemotaxis and that included phosphorylation of Lck, cofilin, PAK1 and extracellular regulated kinase (Erk) and GTP loading of Rac-1 were examined in T cells from DKO mice and found to be normal. These results suggest that WASP and WIP are not essential for T lymphocyte rolling and adhesion, but play important and partially redundant roles in T cell chemotaxis in vitro and homing in vivo and function downstream of small GTPases.

Actins↗

PubMed2005

TACI is mutant in common variable immunodeficiency and IgA deficiency.

The tumor necrosis factor receptor family member TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in B cells. We found that 4 of 19 unrelated individuals with common variable immunodeficiency (CVID) and 1 of 16 individuals with IgA deficiency (IgAD) had a missense mutation in one allele of TNFRSF13B (encoding TACI). One of the four individuals with CVID had a single nucleotide insertion in the other TNFRSF13B allele. None of these mutations were present in 50 healthy subjects. TNFRSF13B mutations cosegregated with the phenotype of CVID or IgAD in family members of four index individuals that we studied. B cells from individuals with TACI mutations expressed TACI but did not produce IgG and IgA in response to the TACI ligand APRIL, probably reflecting impaired isotype switching. These results suggest that TACI mutations can result in CVID and IgAD.

B-Lymphocytes↗

PubMed2005

Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2005

TACI and BAFF-R mediate isotype switching in B cells.

The tumor necrosis factor family members BAFF and APRIL induce Ig isotype switching in human B cells. We analyzed the ability of BAFF and APRIL to induce isotype switching in murine B cells to IgG1, IgA, and IgE. APRIL and BAFF each engage two receptors, transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), on B cells. In addition, BAFF engages a third receptor on B cells, BAFF-R. To determine the role of these receptors in isotype switching, we examined B cells from mice deficient in TACI, BCMA, and BAFF-R. The results obtained indicate that both TACI and BAFF-R are able to transduce signals that result in isotype switching.

Animals↗

PubMed2005

Jun N-terminal kinase is essential for CD40-mediated IgE class switching in B cells.

BACKGROUND: CD40 ligation activates nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases p38 and C-Jun N-terminal kinase (JNK) and causes immunoglobulin class-switch recombination (CSR) in B cells. Both NF-kappaB and p38 are important for CD40-mediated CSR. The role of JNK activation in CD40-mediated isotype switching is unknown. OBJECTIVE: We sought to determine the role of JNK activation in CD40-mediated isotype switching. METHODS: Splenic B cells from BALB/c mice were stimulated with anti-CD40 mAb and IL-4 or with soluble CD40 ligand in the presence or absence of SP600125, an anthrapyrazolone inhibitor of JNK. The following events were examined: IgE production by means of ELISA; S(mu)-S(epsilon) deletional switch recombination by means of digestion circularization PCR; Cepsilon germline, mature epsilon, and activation-induced deaminase (AID) transcription by means of RT-PCR; and proliferation by tritiated thymidine incorporation and surface expression of CD23, CD54, and CD86 by means of FACS analysis. RESULTS: SP600125 at 10 microM drastically inhibited JNK phosphorylation but had little effect on CD40-mediated p38 phosphorylation and expression of the NF-kappaB dependent genes c-Myc and bcl-xL. SP600125 inhibited IgE synthesis by approximately 88% but had no effect on B-cell proliferation and survival in response to anti-CD40 + IL-4 or on upregulation of CD23, CD54, and CD86 in response to CD40 ligation. Analysis of molecular events involved in IgE class switching revealed that SP600125 had no effect on the expression of C(epsilon) germline and AID transcripts. In contrast, SP600125 severely reduced S(mu)-S(epsilon) switch recombination and expression of mature epsilon transcripts. CONCLUSION: These results demonstrate that JNK activation is essential for CD40-mediated CSR to IgE and suggest that JNK is important for AID activity in B cells.

Animals↗