Receptor-like tyrosine phosphatases CD45 and CD148 have distinct functions in chemoattractant-mediated neutrophil migration and response to S. aureus

Abstract

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.

Figure 1. Distinct functions of CD45 and CD148 in bacterial clearance

(A) Mortality curves of mice of the indicated genotypes are shown, where n = 10 for each genotype. DKO refers to Ptprc^−/− Ptprj^TM−/TM− double mutant mice. DKO** were DKO mice treated with food tablets containing amoxicillin (3 mg), Flagyl (0.69 mg) and bismouth (1.185 mg) (1 tablet/cage/week). The difference between DKO and DKO** group is statistically significant (p<0.0001). (B) Animals of indicated genotypes were infected with S. aureus in the air pouch. After 24hr infection, live bacteria in the air pouch were collected by lavage. Recovered Colony Forming Units (CFU) of S. aureus from each mouse were counted and shown. Each symbol represents an individual mouse. Small horizontal lines indicate the means. Bacterial CFU was normalized between experiments based on their bacterial input. CFU shown is following an initial 10² dilution. (C) Neutrophil accumulation in the air pouch after 24 hr of S. aureus infection. Neutrophil counts were determined as (cell count from infected air pouch) × (percentage of neutrophils, identified as CD11b⁺ and Gr1⁺ by flow cytometry). (D) Neutrophil recruitment 6 hr after injection of heat inactivated S. aureus. (C,D) Data are presented as mean ± SEM; p values were calculated by the student t test. For (B) and (C), n=16 wild type mice, n=5 Ptprc^−/− mice, n=12 Ptprj^TM−/TM− mice, n=5 DKO mice were used for experiments. For (D), n=9 wild type mice, n=8 Ptprc^−/− mice, n=9 Ptprj^TM−/TM− mice, n=6 DKO mice were used for experiments.

Figure 2. CD45 and CD148 redundantly regulate neutrophil adhesion, phagocytosis, superoxide production and bacterial killing

(A) Fluorescently labeled BM PMNs from indicated mice were plated on pRGD-coated wells and allowed to adhere at 37°C for 15 min, then exposed to a series of washes in a static adhesion assay. The decrease in fluorescence corresponding to the decrease in cell number was measured and plotted over the series of washes. Error bars represent ± SEM of triplicate samples. (B) Phagocytosis was determined by flow cytometric analysis of FITC-labeled heat inactivated opsonized S. aureus that were phagocytosed by neutrophils purified from mice of the indicated genotypes. Phagocytic units were calculated as (MFI of FITC+ neutrophils) X (% of FITC+ neutrophils). The data points shown here were determined 15 min. after bacteria incubation. Data were pooled from six independent experiments. The graph shows mean ± SEM. (C) Purified neutrophils were plated on microtiter wells in cytochrome c containing media. Production of superoxide by a respiratory burst following 1μm fMLF treatment was measured as the reduction of cytochrome c. Data were pooled from three independent experiments. (D) Intracellular bacterial killing capacity was assessed by viable S. aureus colony forming units (CFU). Percentage of killing was calculated as (1-(remaining CFU/input CFU)) X 100. Data were pooled from three independent experiments.

Figure 3. CD45 and CD148 regulate neutrophil chemotaxis in a distinct ways

(A) Center-zeroed tracks of individual neutrophils in an EZ-Taxiscan chamber migrating towards reservoirs, located at the top of the diagrams, containing 3 μM fMLF. The scales of each graph are equal in μm. (B) Statistical analysis for the EZ-Taxiscan migration assay. Displacement rate (μm min⁻¹), track velocity (μm min⁻¹) and directionality (ratio of displacement to track, value 1 as maximum) were calculated by Volocity^™. Each symbol represents an individual cell; small horizontal lines indicate means. p values were calculated by the student t test. See also Fig. S1, Movie M1,M2,M3,M4.

Figure 4. CD45 and CD148 differentially regulate fMLP mediated increase of intracellular Ca²⁺ in dose dependent manner

Purified neutrophils from mice of the indicated genotypes were loaded with Fluo3-AM and Fura Red, then intracellular free-Ca²⁺ concentrations were monitored before and after addition of different dose of fMLF (A), (B), (C) or ionomycin (D). Data are representative of three independent experiments.

Figure 5. CD45 and CD148 differentially regulate signaling events following fMLF stimulation

(A) BM cells from mice of the indicated genotypes were stimulated with fMLF (0.2 μM) for indicated time, fixed, permeabilized, and then stained with phospho-Akt along with other surface makers. Neutrophils were identified as CD11b⁺Gr1⁺. Phospho-Akt of stimulated (2.5 min, shown as black lines) and control samples (shown as filled histograms) were overlaid. Percentages of pAkt⁺ cells over a time course are shown on the right side. Data are representative of three independent experiments. (B) BM cells were treated same as in (A) and phospho-ERK was stained similarly. Percentages of pERK⁺ cells over a time course are shown on the right side. Data are representative of three independent experiments.

Figure 6. Disruption of SFKs results in impairment on fMLF induced signaling

(A) Purified neutrophils from mice of the indicated genotypes were loaded with Fluo3-AM and Fura Red, and [Ca²⁺]_i concentrations were monitored before and after addition of 0.2 μm fMLF. (B) Wild-type neutrophils were treated similarly as in (A), however prior to addition of fMLF, cells were incubated with or without 10 μm PP2 for 3 minutes. (C) (D) Neutrophils from wild-type, Lyn^−/− (C), or wild-type, Hck^−/−Fgr^−/− (D) mice were treated similarly as in (A). (E) BM cells from mice of the indicated genotypes were stimulated with fMLF (0.5 μM) for the indicated times, fixed, permeabilized, and then stained with phospho-ERK along with other surface makers. Neutrophils were identified as CD11b⁺Gr1⁺. Phospho-ERK of stimulated (1 min) and control samples were overlaid as histograms (control as grey shade and stimulated as black line). Percentages of pERK⁺ cells over a time course are shown on the right side. Data are representative of three independent experiments. (F) fMLF mediated chemotaxis was measured on an EZ-Taxiscan chamber as shown in Fig. 3. Left panel shows center-zeroed tracks of individual neutrophils on migrating towards reservoirs, located at the top of the diagrams, containing 0.5 μM fMLF. The scale of each graph is equal in μm. Right panel is a statistical analysis for the EZ-Taxiscan migration assay. p values were calculated by student t test. See also Fig. S3, Movie M5, M6.

Figure 7

(A) BM cells (40×10⁶/sample) were harvested and then lysed. Hck or Lyn was immunoprecipitated and subject to immunoblotting with antiphosphotyrosine (pTyr) Abs or anti-Hck and Lyn for loading controls. Specificities of Hck and Lyn Abs were confirmed with whole cell lysates from Hck^−/− or Lyn^−/− mice. Quantification of inhibitory tyrosine phosphorylation amounts were normalized to loading controls and the fold changes relative to wild type were graphed. For Hck n=3 and for Lyn n=4. (B) Neutrophils (15×10⁶/sample) were treated with fMLP (3μm) for indicated times and then lysed. SHP-1 was immunoprecipitated and subjected to immunoblotting with antiphosphotyrosine (pTyr) Ab 4G10 or SHP-1 as a loading control. Data are representative of three independent experiments. See also Fig. S4.