PMN oxidative burst—Ca²⁺ chelation.

<p>Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized <i>E</i>. <i>coli</i> (1-2x10⁹/ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the PMN population. (A) BAPTA-AM treatment cause a stronger reduction of <i>E</i>. <i>coli</i>-induced oxidative burst in HD than XLA (▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as <i>p</i> value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. <b>(</b>B and C) It shown PMN <i>E</i>. <i>coli-</i>induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown PMN PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).</p

Monocytes and oxidative burst in XLA patients and HD.

<p>The quantitative evaluation of leukocyte oxidative burst was observed by incubating whole blood samples in a water bath at 37°C with opsonized <i>E</i>. <i>coli</i> (1-2x10⁹/ml) or PMA (1.62mM). The conversion of DHR 123 to R 123 were used to evaluated the intracellular ROS production. (A and B) XLA patients show a similar monocytes <i>E</i>. <i>coli</i>-induced and PMA-induced oxidative burst compared to HD. (C and D) XLA patients show a comparable PMN <i>E</i>. <i>coli</i>-induced and PMA-induced oxidative burst to HD. After IVIg, monocyte <i>E</i>. <i>coli</i>-induced oxidative burst slightly decreased (▪p = 0.004), but it remain unaltered when PMA was provided. After IVIg, PMN ROS production remain unaltered. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, determined by the nonparametric Wilcoxon Signed Rank test, is indicated as <i>p</i> value.</p

Monocytes oxidative burst—Ca²⁺ chelation.

<p>Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized <i>E</i>. <i>coli</i> (1-2x10⁹/ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. (A) BAPTA-AM treatment cause a stronger reduction of <i>E</i>. <i>coli</i>-induced oxidative burst in HD than XLA <i>(</i>▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as p value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. (B and C) It shown monocytes <i>E</i>. <i>coli</i>-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown monocytes PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).</p

The lack of BTK does not impair monocytes and polymorphonuclear cells functions in X-linked agammaglobulinemia under treatment with intravenous immunoglobulin replacement

<div><p>The lack of BTK in X-linked agammaglobulinemia (XLA) patients does not affect monocytes and polymorphonuclear cells (PMN) phenotype and functions. In this study, we show that XLA patients had an increased frequency of the intermediate monocytes subset and that BTK-deficient monocytes and PMN had a normal expression of receptors involved in the activation and cellular responses. We demonstrate that BTK is not required for migration, phagocytosis and the production of reactive oxygen species (ROS) following engagement of FC gamma receptors (FcγR). XLA monocytes and PMN showed an efficient calcium (Ca²⁺)-independent activation of oxidative burst, suggesting that oxidative burst is less dependent by Ca²⁺ mobilization. The phagocytosis was functional and it remained unaltered also after Ca²⁺ chelation, confirming the independence of phagocytosis on Ca²⁺ mobilization. Intravenous immunoglobulin (IVIg) infusion exerted an anti-inflammatory effect by reducing the frequency of pro-inflammatory monocytes. In monocytes, the IVIg reduce the oxidative burst and phagocytosis even if these functions remained efficient.</p></div

Schematic representation of signaling pathways for phagocytosis and oxidative burst.

<p>As a result of FcγR clustering, two separate pathways lead to phagocytosis, through a calcium independent way, or to the oxidative burst in a calcium dependent manner. Syk induce the activation of Vav and the downstream signaling that lead to the phagocytosis process. The main pathway for the activation of the oxidative burst consist in the activation of Btk by Syk and the consequent phosphorylation of PLCγ2. PLCγ2 activated produce DAG and IP3 from PIP3. DAG directly activate PKC while IP3 bind IP3R on endoplasmic reticulum inducing Ca²⁺ mobilization that lead to PKC activation and finally to the oxidative burst. The lack of BTK in XLA patients is effectively bypassed by the direct stimulation of PLCγ2 by Syk. The inhibition of Ca²⁺ mobilization by BAPTA-AM strongly reduce the oxidative burst. PMA is able to bind PKC to the same binding site of DAG and bypass both the production of DAG and the calcium flux from endoplasmic reticulum.</p

Plasma levels of IL-8 in XLA patients and HD.

<p>IL-8 production was measured by using a kit ELISA. IL-8 plasma levels were similar in XLA and HD. (A) After <i>E</i>. <i>Coli</i> stimulation, IL-8 increased in XLA and HD. (B) After IVIg infusion, IL-8 levels remained unchanged and the level after <i>E</i>. <i>coli</i> stimulation was well preserved. Data show that only one XLA patient (gold) with a severe inflammatory condition had a very high IL-8 plasma levels. The same patient exhibited high level of IL-8 after <i>E</i>. <i>Coli</i> stimulation. Concentrations were expressed as pg/ml. <i>*</i>p = 0.042; **p = 0.043.</p

Monocytes and PMN phagocytosis in XLA patients and HD.

<p>The quantitative determination of leukocyte phagocytosis was tested by incubating whole blood samples in a water bath for 10 min at 37°C with FITC-labeled <i>E</i>. <i>coli</i> (2x10⁹/ml). The percentage of cells having performed phagocytosis was analyzed as number of ingested bacteria and results were expressed as Mean Fluorescence Intensity. XLA patients show a similar monocytes phagocytosis compared to HD. (A) After IVIg administration monocytes phagocytosis decreased (▪p = 0.04). XLA patients show a similar PMN phagocytosis compared to HD. (B) After IVIg administration PMN phagocytosis remains unchanged. Histograms denote mean values and bars standard deviation. Statistical significance, determined by the nonparametric Wilcoxon Signed Rank test, is indicated as <i>p</i> value.</p

PMN migration.

<p>The quantitative determination of PMN chemotaxis was observed by incubating heparinized whole blood samples on leukocyte separation medium for isolation of leukocyte-rich plasma (LRP). After isolation, LRP were incubated in a water bath for 30 min at 37°C with fLMP. Values are expressed as number of PMN which have migrated by flow cytometry. (A) Data show that PMN migration induced by fMLP was comparable in XLA patients and HD. (A) After IVIg, the PMN migration remained unaltered. Histograms denote mean values and bars standard deviation. (B, C and D) An example of the FACS analysis of PMN migration of a representative XLA patient before and after IVIg infusion. The migrated cells were gated and analyzed within the quadrants of the dot plots.</p

CD181, CD11b, CD11c and Siglec 9 expression on monocytes subsets from HD and XLA patients before and after IVIg infusion.

<p>Whole blood samples were analyzed for the expression of CD181, CD11b, CD11c and Siglec 9 before and after IVIg infusion. The expression of all surface receptors was evaluated by performing a staining at 4°C for 30 min with specific fluorochrome-labeled antibody. Samples were washed, suspended in ice-cold PBS and analyzed by flow cytometry. XLA patients show a similar CD181, CD11b, CD11c and Siglec 9 expression on monocytes subsets compared to HD. After IVIg infusion the expression of all receptors on monocytes subsets remained unaltered. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation.</p

Frequencies of non classical, intermediate and classical monocytes increased from XLA patients before and after IVIg infusion respect to HD.

<p>(A and B) Monocytes subpopulations were phenotypically classified according to their expression of CD14 and CD16 into classical (CD14⁺⁺CD16^-), intermediate (CD14⁺⁺CD16⁺) and non classical monocytes (CD14⁺CD16⁺⁺) in a representative XLA patient before and after IVIg infusion. Percentages denote mean values. (C) Histograms show that non classical and classical monocyte frequencies from XLA patients are similar to that observed on HD and that IVIg infusion did not change their frequency. Intermediate monocytes percentage is increased in XLA patients (▪p = 0.01), IVIg infusion induce their reduction (▪▪p = 0.04) even if remained at higher level respect to HD (▪▪▪p = 0.01). (D) Histograms denote the percentage of increase of the three monocytes subsets of XLA patients respect to HD. Histograms denote mean values and bars standard deviation. Statistical significance as determined by the nonparametric Mann Whitney and Wilcoxon Signed Rank test is indicated as <i>p</i> value.</p