Bruton's tyrosine kinase is not essential for LPS-induced activation of human monocytes

Bruton’s tyrosine kinase is not essential for LPS-induced activation of human monocytes Rebeca Pe´rez de Diego, PhD,a Eduardo Lo´pez-Granados, MD, PhD,a Maite Pozo, PhD,b Cristina Rodrı´guez, PhD,b Prado Sabina, PhD,a Antonio Ferreira, MD, PhD,a Gumersindo Fontan, MD, PhD,a Maria Cruz Garcı´a-Rodrı´guez, MD, PhD,a and Susana Alemany, MD, PhDb Madrid, Spain

Background: X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton’s tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. Objective: The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. Methods: Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-a and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. Results: We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-a and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. Conclusion: Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. Clinical implications: These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models. (J Allergy Clin Immunol 2006;117:1462-9.)

Basic and clinical immunology

From athe Immunology Unit, University Hospital ‘‘La Paz,’’ and bInstituto de Investigaciones Biome´dicas, CSIC, Medical Faculty, Autonomic University of Madrid. This work was supported by ‘‘Fondo de Investigacio´n Sanitaria (FIS)’’ grant no. 020982, by the CICYT (BMC2002-00437), and by AICR and Fundacio´n ‘‘La Caixa.’’ RPdD is the recipient of a fellowship from the FIS, CR from the ME, and MP from the AICR. Disclosure of potential conflict of interest: The authors have declared they have no conflict of interest. Received for publication November 21, 2005; revised January 18, 2006; accepted for publication January 24, 2006. Available online March 31, 2006. Reprint requests: Rebeca Pe´rez de Diego, PhD, Po Castellana, 261, Immunology Unit, University Hospital La Paz, 28046 Madrid, Spain. E-mail: rperez. [email protected]; [email protected]. 0091-6749/$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2006.01.037

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Key words: X-linked agammaglobulinemia, Bruton’s tyrosine kinase, LPS signaling, mitogen-activated protein kinases, tumor necrosis factor a, IL-6, oxidative burst activity, monocytes

Mutations in the Bruton’s tyrosine kinase (Btk) gene (BTK) are responsible for human X-linked agammaglobulinemia (XLA).1,2 Btk is a member of the Tec family of kinases, which are essential for early human B-cell differentiation. Patients with XLA present a lack of circulating B cells, severe hypogammaglobulinemia, and impaired antibody responses and consequently exhibit increased susceptibility to bacterial and enteroviral infections.3,4 The natural model of XLA, the X-linked immunodeficiency (Xid) mouse, which is caused by a missense mutation in the N-terminal part of the murine Btk, show a phenotype resembling the human disease but with less severity. Indeed, Xid mice present a 50% decrease in circulating B cells, low levels of IgM and IgG3, and the capacity to mount T cell–dependent antibody responses.4,5 Btk is expressed in all the hematopoietic lines, except for T cells and plasma cells,6 and different extracellular signals, including erythropoietin, stem-cell factor, Fc receptors, ILs, and LPS, also lead to Btk activation in different cell types.7-10 LPS is a major component in the cell wall of gramnegative bacteria and a potent inductor of responses in the myeloid lineage. LPS binding to the CD14–Toll-like receptor 4 (TLR4) complex triggers an intracellular signaling cascade that involves the recruitment of several proteins, including MyD88, Mal, IL-1 receptor–associated kinase (IRAK), and TNF receptor–associated factor (TRAF) 6, to the receptor complex, resulting in the activation of the TGF-B–activated kinase (TAK1).11,12 TAK1 acts as a common activator of the transcription factor nuclear factor kB and the mitogen-activated protein (MAP) kinases p38 and c-Jun amino-terminal kinase (JNK).12 The activation of nuclear factor kB, as a consequence of the IkBa degradation, and the activation of the different MAP kinases by LPS mediate the secretion of TNF-a and IL-6, essential cytokines of the inflammatory reaction in monocytes.13,14 Immunoprecipitation experiments performed in the human promonocytic cell line THP-1 have demonstrated that Btk interacts with TLR4, Myd88, Mal1, and IRAK4 (for a review, see Jefferies and O’Neill15). In addition, Btk activation and enhanced TNF-a production with Btk overexpression occur in LPS-stimulated human monocytes.16 The phagocytosis and intracellular reactive oxygen-dependent killing (oxidative burst) of pathogens by activated myeloid cells is

Abbreviations used BCR: B-cell receptor Btk: Bruton’s tyrosine kinase DC: Dendritic cell Erk: Extracellular signal–regulated kinase FITC: Fluorescein isothiocyanate IRAK: IL-1 receptor–associated kinase JNK: c-Jun amino-terminal kinase MAP kinase: Mitogen-activated protein kinase PMA: Phorbol 12-myristate 13-acetate PR: Phycoerythrin ROI: Reactive oxygen species TLR4: CD14/Toll-like receptor 4 Xid: X-linked immunodeficiency XLA: X-linked agammaglobulinemia

an essential component of the host’s response to bacterial and fungal infections.17 Defective LPS responses in myeloid cells, as well as a defective respiratory oxidative burst in macrophages and neutrophils and reduced severity of induced inflammatory disease models, have been reported in the Xid mouse.18 Altogether, these findings suggest a participation of Btk in different events of the myeloid line activation but simultaneously raise the question of the nonredundant need for Btk in human myeloid cells. A previous report showed that LPS-driven differentiation of XLA monocytes to dendritic cells (DCs) is conserved,19 and patients with XLA do not present poor inflammatory responses, the typical infections associated with defective intracellular killing caused by myeloid defects in other primary immunodeficiencies, or both. In this article we explore the functional relevance of the lack of Btk protein in human monocytes. Our results show that although Btk might play a role in LPS signaling in human monocytes, the major early signaling molecular events, such as oxidative burst, early TNF-a and IL-6 production, and JNK, p38, and extracellular signal–regulated kinase (Erk) 1/Erk2 activation, were found to be equal to those from the same number of healthy age- and sexmatched control subjects. Moreover, these data indicate that although Btk might play a role in LPS signaling in human monocytes, major early signaling molecular events are conserved in Btk-null monocytes, suggesting a functional redundancy of tyrosine kinases in early LPS signaling.

METHODS Subjects Seven unrelated patients with XLA, determined according to the criteria of the IUIS Scientific Group,20 and 7 healthy age-matched control subjects were included in this study. The ethics committee approved the protocol, and written consent was obtained before blood was drawn. Samples were collected immediately before a new routine intravenous immunoglobulin dose and without any evidence of infection in the patients.

BTK mutation and Btk expression studies BTK genetic study was performed by means of PCR and sequencing, as previously described.21 For Btk expression studies,

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PBMCs were isolated on a lymphocyte separation gradient centrifugation (Ficoll Hypaque; Sigma, St Louis, Mo). Intracellular Btk expression was analyzed with a modification of the protocol of Futatani et al.22 PBMCs (5 3 105) were fixed and permeabilized (FIX and PERM; Caltag, Burlingame, Calif) and washed in PBS, 5% FCS, 1.5% BSA, and 0.0055% EDTA. Cells were incubated with anti-CD14-phycoerythrin (anti-CD14–PE; Becton Dickinson, San Jose, Calif) and with either an IgG1 anti-Btk (48-2H) mAb (provided by Dr Miyawaki, Toyama University, Japan) or an IgG1 isotype control (Becton Dickinson). Goat anti-mouse IgG1– fluorescein isothiocyanate (IgG1–FITC; Southern Biotechnology, Birmingham, Ala) was used as a secondary antibody. Intracellular Btk expression was analyzed in monocytes selected by means of size, granularity, and CD14-positive staining in a FACScalibur with the Cell Quest software (Becton Dickinson). Btk detected by means of Western blot analysis was performed by using PBMCs as described previously23 with anti-Btk (48-2H) followed by a secondary peroxidase goat anti-mouse antibody (Sigma). Membranes were reblotted with anti-b-actin (Sigma) as a control of protein loading. Antibody binding was revealed with the chemiluminescent ECL method (Amersham-Pharmacia-Biotec, Buckinghamshire, United Kingdom).

Determination of Erk1/Erk2, JNK, and p38 phosphorylation MAP kinase activation was analyzed in PBMCs (1 3 106 cells/ mL) after stimulation for 20 minutes with LPS (500 ng/mL, Sigma) or for 15 minutes with phorbol 12-myristate 13-acetate (PMA; 4 mg/mL, Sigma), as described previously.23 Monocytes were selected on the basis of size, granularity, and CD14-positive staining. Intracellular staining of the different phospho-MAP kinases was performed as described above for Btk by using phospho-Erk1/2 (Santa Cruz Biotechnology, Santa Cruz, Calif) and (pT138/pY185) phospho-specific JNK antibodies, followed by an anti-mouse IgG1FITC antibody (Southern Biotechnology). For p38, an anti-phosphoT180/Y182-p38-PE (Becton Dickinson) and an anti-IgG1-PE isotype negative control (Becton Dickinson) were used. For MAP kinase Western blotting studies, cell extraction, protein separation, and membrane blotting were performed as described above, except that phospho-Erk1/2 (Santa Cruz Biotechnology) or phospho-p38 (Cell Signalling Technology, Beverly, Mass) primary antibodies were used.

Intracellular flow cytometric analysis of TNF-a and IL-6 production PBMCs (1 3 106 cells/mL) were stimulated with LPS (at 0, 1, and 10 ng/mL) for 4 hours in the presence of 10 ng/mL Brefeldin A (Sigma) to inhibit cytokine secretion. Cells were then washed with PBS–1% BSA, fixed, and permeabilized (FIX and PERM, Caltag). Monocytes were selected from the PBMC population by means of forward scatter characteristics (FSC)-height/side scatter characteristics (SSC)-height, and the intracellular cytokine production was measured after staining with anti-CD14-FITC (Becton Dickinson) and anti-TNF-a–PE or anti-IL-6–PE (Becton Dickinson) in the CD14 positive staining. Anti-IgG2b–FITC (Becton Dickinson) and anti-IgG1–PE (Becton Dickinson) were used as isotype-negative controls.

Quantification of the oxidative burst activity of monocytes and granulocytes Whole blood from patients and healthy control subjects was stimulated with heat-inactivated Escherichia coli (1.6 3 108/mL) or with PMA (1.35 mM) for 10 minutes at 37°C. The oxidative burst activity measured on the basis of reactive oxygen species (ROI) production was determined in monocytes and polymorphonuclear cells by

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TABLE I. Clinical and molecular data from patients with XLA Patient no.

Age at IgG, IgA, IgM, IgE, B diag- mg/ mg/ mg/ UI/ Cell, dL mL % dL dL nosis

1 2 3 4

3y <7 5 3y 55 7 9 mo <200 <6 4y 76 <4

<4 20 <4 3

5 6

1y 11 mo

201 9 <33 <6

28 <4

7

4 mo

139 <6

6

<2 2 <2 <2

Mutation

0 R13X 0.06 A207X216 0 G613A 0 V131GS174 <2 0 K218X228 <2 0 INT13GG/CT 4 0.5 DEL 59

Btk expression

Null Null Null Null Null Null Null

IgG, IgA, and IgM levels at diagnosis B, and percentage of B cells in the total lymphocyte population are shown. The mutation in the protein sequence is shown.

means of flow cytometry with the Phagoburst kit (Orpegen Pharma, Heidelberg, Germany), according to the manufacturer’s instructions.

RESULTS Analysis of Btk expression in patients with XLA Previous studies suggested Btk participation in some intracellular signaling events triggered by LPS in myeloid cells.15,16 We sought to analyze these events in circulating monocytes from 7 unrelated patients with XLA in whom a null Btk protein expression had been previously confirmed. Clinically, all presented a classic phenotype with severe hypogammaglobulinemia, recurrent bacterial infections, and an onset of the disease early in life. The genetic study showed different mutations in the BTK coding sequence (Table I). The absence of Btk expression was demonstrated by means of Western blotting analysis (Fig 1, A), as well as by means of intracellular flow cytometric analysis (Fig 1, B).

Basic and clinical immunology

Early LPS signaling induces phosphorylation of MAP kinases in Btk-null human monocytes A similar increase in the phosphorylation state of 3 main MAP kinases, Erk1/Erk2, JNK, and p38, measured on the basis of the intracellular fluorescence intensity, was observed when PBMCs from 7 Btk-null patients with XLA and from 7 healthy control subjects were stimulated with LPS for 20 minutes (Fig 1, C-F). The phosphorylation increment of Erk1/Erk2, p38, and JNK in monocytes in response to LPS was totally similar between the mean of the 7 patients and the mean of the 7 healthy control subjects (Fig 1, F). In fact, an unpaired 2-tailed t test using a CI of 0.95 of the data resulted in a P value of .3358 for phospho-Erk1/Erk2, a P value of .6374 for phospho-JNK, and a P value of .2434 for phospho-p38. As expected, similar results were obtained when phosphoErk1/Erk2 and phospho-p38 levels from LPS-stimulated monocytes from Btk-null patients with XLA and from healthy control subjects were determined by means of

Western blot analysis (Fig 1, A). On the other hand, IkBa protein is also degraded in Btk-null XLA monocytes in response to a 15-minute LPS stimulation (data not shown).

TNF-a and IL-6 production in Btk-null human monocytes The activation of the 3 MAP kinase pathways, as well as the degradation of IkBa, are essential events to produce the different cytokines in response to LPS signaling in human monocytes. We next decided to analyze TNF-a and IL-6 synthesis in LPS-activated monocytes. To ensure that synthesis of these cytokines was due to activation of the intracellular signaling by LPS and not due to an extracellular mediator secreted by LPS stimulation, we measured in Btk-null and normal monocytes the TNF-a and IL-6 cytoplasmic accumulation after 4 hours of stimulation with LPS in the presence of a protein secretion inhibitor. Production of both cytokines was fully comparable in the XLA and the healthy donor groups (Fig 2), and statistical analysis of the data did not reveal any variability between the groups (data not shown). In agreement with these data, similar TNF-a and IL-6 mRNA levels were obtained by means of RT-PCR real-time analysis in monocytes from patients with XLA and healthy control subjects after LPS stimulation (data not shown). Respiratory burst activity in Btk-null monocytes and neutrophils The oxidative burst of pathogens by means of activated myeloid cells is an essential component of the host’s response to bacterial and fungal infection. We analyzed the capacity of ROI generation in activated Btk-null human monocytes by measuring the intracellular fluorescent intensity emitted by the dye dihydrorhodamine 123 (burst test). Peripheral circulating monocytes from the 7 patients with XLA and the 7 healthy control subjects presented a similar burst activity in response to heatinactivated E coli or PMA (Fig 3, A). Similar results were obtained when the neutrophil population was compared (Fig 3, B). As expected, statistical analysis of the data obtained between the XLA and control groups in different conditions did not reveal any difference (data not shown). In agreement with the lack of clinical evidence of intracellular killing deficiencies observed in patients with XLA, these data indicate that the oxidative burst induction in human myeloid cells is not dependent on Btk expression. DISCUSSION The main immune defect observed in patients with XLA is the lack of mature B cells, and the clinical picture and spectrum of infections is adequately justified just by the absence of antibody-mediated responses. XLA is the prototypic antibody immunodeficiency caused by a block of B-cell differentiation in the bone marrow as a result of mutations in the BTK gene.2,3 These data provide evidence of the importance of Btk in the intracellular signaling initiated by the B-cell receptor (BCR). In response to

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BCR activation, Btk, the only member of the Tec family expressed in B cells, translocates to the membrane, where it becomes activated, playing a key role in the expansion and fine tuning of BCR signaling.24 Moreover, Btk-deficient B cells have an impaired Erk1/Erk2 and JNK MAP kinase activation after BCR engagement.25 Btk is also activated in response to LPS stimulation, although the exact mechanism is still unknown.15 According to the recent implications of Btk in LPS signaling15,16 and the fact that defective monocyte responses are not evident in patients with XLA, we decided to analyze the integrity of LPS-induced events in Btk-null human monocytes. We found that on LPS stimulation, the activation of the 3 major MAP kinases increases similarly in Btk-null and control monocytes. These data clearly demonstrate that Btk plays a different role in the activation of the MAP kinase pathways in response to either BCR

or TLR4. Btk is the only member of the Tec family expressed in B cells; however, another tyrosine kinase might compensate the lack of Btk in monocytes, allowing a rapid MAP kinase phosphorylation in response to LPS. We analyzed the intracellular TNF-a production on 4 hours of LPS stimulation in Btk-null monocytes and again found it similar to that seen in normal cells. However, a recent publication reported that an overexpression of Btk in human monocytes synergizes with LPS in the TNF-a production by means of its mRNA stabilization.16 Our results clearly show that although Btk participates in LPS signaling, it is not essential in the early production of TNF-a. Therefore it is possible that overexpressed Btk upregulates signal transduction pathways that physiologic levels of Btk do not. In the previously mentioned report, XLA monocytes separated by means of plastic adherence showed a 50% decrease in TNF-a production after 18

Basic and clinical immunology

FIG 1. LPS activation of MAP kinases and BTK expression in XLA monocytes. A, Representative Western blot analysis of Btk, phospho-ERK, and phospho-p38 expression in XLA and control PBMCs. B through E, Representative example of Btk (Fig 1, B), phospho-Erk1/2 (Fig 1, C), phospho-p38 (Fig 1, D), and phospho-JNK (Fig 1, E) intracellular staining in Btk-null and control monocytes. Thick line, Specific anti-Btk or anti-phosphoprotein staining; dashed line, IgG1 negative control; thin line, secondary antibody alone. Specific signal: D5ðMean fluorescence with the specific antibody Þ2ðMean fluorescence of the isotype contolÞ. F, Intensity of phospho-specific MAP kinase staining on LPS activation in XLA and control monocytes measured as the mean fluorescent intensity obtained in the intracytoplasmic flow cytometric analysis with the different phospho-specific MAP kinase antibodies in response to LPS. The value represents ðMean fluorescent intensity of LPS
stimulated cellsÞ2ðMean fluorescent intensity of unstimulated cellsÞ for each patient and healthy control subject analyzed in parallel.

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FIG 2. Intracellular TNF-a and IL-6 accumulation in Btk-null and control monocytes in response to LPS. One representative example of the intracellular TNF-a and IL-6 monocyte production in response to different concentrations of LPS performed in the 7 patients with XLA and control subjects is shown. The percentage of positive cells in the total monocyte population is represented. A, Btk-null monocytes from a patient with XLA. B, Healthy control subject.

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FIG 3. Respiratory burst activity in Btk-null and control monocytes and polymorphonuclear cells. Representative example of 1 of 7 ROI productions analyzed by means of flow cytometry in monocytes (A) or polymorphonuclear cells (B) from Btk-null patients and healthy control subjects. The figure represents the ethidium bromide (BrEt2)–positive (alive cells) versus dihydrorhodamine 123 oxidation (ROI). Negative, Nonstimulated cells; PMA, stimulated with PMA; E. coli, stimulated with E coli. The percentage of double-positive cells is indicated.

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hours of LPS stimulation16; however, the type of mutation and the absence or not of Btk expression in the XLA monocytes were not stated. Additionally, it should be noted that a defective TNF-a production in response to LPS is clinically evidenced by a poor inflammatory reaction, with defective acute-phase responses. This is the main clinical feature in IRAK4 deficiency, a key protein in TLR4 signaling.26 In contrast, these findings are not part of the XLA phenotype, as should be expected if Btk was essential in LPS signaling. In addition, previous experimental evidence of normal LPS responses in XLA myeloid cells has been reported, XLA monocyte precursors differentiate normally to DCs in response to LPS, as assessed on the basis of de novo expression of CD83, normal IL-12 and IL-10 production, and upregulation of MHC class II, B7.1, and B7.2 molecules. LPS-stimulated XLA DCs acquire the ability to prime naive T cells as control cells. These data indicate that Btk is not involved in LPS-driven DC differentiation and maturation and that XLA DCs can act as fully competent antigen-presenting cells in T cell–mediated immune responses.19 All these data indicate that the dependency, mechanism, and exact timing of the Btk participation in LPS signaling in human myeloid cells needs further study. The intracellular killing based on oxidative burst activity of neutrophils and monocytes is an important component of host defense against intracellular bacterial and fungal infections. Recently, different studies from the same authors have shown a defective burst of ROIs in Xid macrophages and neutrophils.18,27 We analyzed the burst activity of Btk-null XLA monocytes and neutrophils in response to LPS (data not shown), E coli, and PMA and found it to be similar when compared with that seen in healthy control subjects. In human subjects molecular defects in proteins of the reduced nicotinamide adenine dinucleotide phosphate oxidase system, an essential component in the generation of the oxidative burst, are associated with the appearance of granulomas as a consequence of the impaired elimination of intracellular pathogens by neutrophils and monocytes.28 Granuloma formation is not part of the clinical spectrum of XLA,3 suggesting that human Btk is not essential for oxidative burst activity in human myeloid cells. Patients with XLA have recurrent bacterial infections that are well controlled if the immunoglobulin replacement is promptly initiated. Other evidences of impaired immune responses, apart from the antibody-mediated responses, are not part of the XLA phenotype.3 The same authors mentioned above have described decreased numbers of periphery and bone marrow neutrophils in the Xid mouse, suggesting a defect in the murine myeloid line differentiation.18 The question of a possible impairment of myeloid differentiation caused by Btk deficiency has been raised for some years. However, neutropenia is only present in 20% of patients with XLA, and there is no direct evidence of functional impairment in human Btk-deficient neutrophils. Moreover, there is normalization of neutrophil counts with immunoglobulin, the neutropenia is not exclusive of XLA–X-Linked hyper-IgM, and the patients with

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common variable immunodeficiency can also present neutropenia, strongly suggesting that this cytopenia is a secondary cause and not a direct consequence of Btk deficiency.29 Additionally, regarding the need of Btk for myeloid differentiation, Btk detection in monocytes is a diagnostic assay in female carriers in whom Btk-null monocytes are present in peripheral blood and do not show a selective proliferate disadvantage in comparison with cells expressing the normal allele. The discrepancies between the results in the Xid mouse, extensively used as a model in the study of Btk function in different cell types, and the patients with XLA could be related with differences in the Btk dependence in myeloid cells from both species. The Xid phenotype is less severe than human XLA, and this difference is not related with the R28C mutation itself because the phenotype of the Btk knockout mouse is similar to that of the Xid mouse and not to that of the human patient with XLA. In addition, patients with an XLA phenotype and a similar R28C mutation have been identified. The discrepancies between the human and the mouse disease suggest different B-cell dependency of Btk in both species. Our results suggest that previous data on the involvement of Btk in myeloid activities in the Xid mouse cannot be fully extrapolated to human monocytes. In conclusion, we have provided clear evidence that LPS-induced oxidative burst, early TNF-a and IL-6 production, and Erk1/Erk2, JNK, and p38 phosphorylation are not affected by the complete absence of Btk in human monocytes. These data are in accordance with the clinical characteristics of XLA, in which no defects in innate immune and inflammatory responses are evident. We thank the participating patients for their invaluable contribution to the project.

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10. Sato S, Katagiri T, Takaki S, Kikuchi Y, Hitoshi Y, Yonehara S, et al. IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton’s tyrosine and Janus 2 kinases. J Exp Med 1994;180:2101-11. 11. Palsson-McDermott EM, O’Neill LA. Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 2004;113:153-62. 12. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4:499-511. 13. Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85-94. 14. Kollias G, Douni E, Kassiotis G, Kontoyiannis D. The function of tumour necrosis factor and receptors in models of multi-organ inflammation, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Ann Rheum Dis 1999;58(suppl 1):I32-9. 15. Jefferies CA, O’Neill LA. Bruton’s tyrosine kinase (Btk)—the critical tyrosine kinase in LPS signalling? Immunol Lett 2004;92:15-22. 16. Horwood NJ, Mahon T, McDaid JP, Campbell J, Mano H, Brennan FM, et al. Bruton’s tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production. J Exp Med 2003;197:1603-11. 17. Sawyer DW, Donowitz GR, Mandell GL. Polymorphonuclear neutrophils: an effective antimicrobial force. Rev Infect Dis 1989;11(suppl 7): S1532-44. 18. Mangla A, Khare A, Vineeth V, Panday NN, Mukhopadhyay A, Ravindran B, et al. Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses. Blood 2004;104:1191-7. 19. Gagliardi MC, Finocchi A, Orlandi P, Cursi L, Cancrini C, Moschese V, et al. Bruton’s tyrosine kinase defect in dendritic cells from X-linked agammaglobulinaemia patients does not influence their differentiation, maturation and antigen-presenting cell function. Clin Exp Immunol 2003;133:115-22.

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20. Conley ME. Diagnostic guidelines—an international consensus document. Clin Immunol 1999;93:189. 21. Rodriguez MC, Granados EL, Cerdan AF, Casariego GF. Molecular analysis of Bruton’s tyrosine kinase gene in Spain. Hum Mutat 2001; 18:84-8. 22. Futatani T, Miyawaki T, Tsukada S, Hashimoto S, Kunikata T, Arai S, et al. Deficient expression of Bruton’s tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood 1998;91: 595-602. 23. Caivano M, Rodriguez C, Cohen P, Alemany S. 15-Deoxi-D-12,14-prostaglandin J2 regulates endogenous Cot MAP kinase kinase kinase 1 activity induced by LPS. J Biol Chem 2003;278:52124-30. 24. Satterthwaite AB, Witte ON. The role of Bruton’s tyrosine kinase in B-cell development and function: a genetic perspective. Immunol Rev 2000;175:120-7. 25. Jiang A, Craxton A, Kurosaki T, Clark EA. Different protein tyrosine kinases are required for B cell antigen receptor-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase 1, and p38 mitogen-activated protein kinase. J Exp Med 1998;188:1297-306. 26. Picard C, Puel A, Bonnet M, Ku CL, Bustamante J, Yang K, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003;299:2076-9. 27. Mukhopadhyay S, Mohanty M, Mangla A, George A, Bal V, Rath S, et al. Macrophage effector functions controlled by Bruton’s tyrosine kinase are more crucial than the cytokine balance of T cell responses for microfilarial clearance. J Immunol 2002;168:2914-21. 28. Johnston RB Jr. Clinical aspects of chronic granulomatous disease. Curr Opin Hematol 2001;8:17-22. 29. Cham B, Bonilla MA, Winkelstein J. Neutropenia associated with primary immunodeficiency syndromes. Semin Hematol 2002;39:107-12.

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