Fig 1.
Slamf8 negatively and Slamf1 positively affects DC migration from the skin to draining lymph nodes after Hapten-FITC sensitization.
(A) Representative dot plots (gated on CD11c+ cells) and (B) percentages of migratory MHC-IIhi FITC+ DCs in the draining LNs of wt and Slamf8-/- mice, 24 h after painting dorsal skin with Hapten-FITC. Total number of migratory (CD11c+MHC-IIhiFITC+) DCs in the draining Lymph Nodes (LNs) of (C) Slamf8-/- mice or (D) Slamf1-/- mice, 24 h after FITC painting. Values represent mean. The data are representative of three or more independent experiments.
Fig 2.
Slamf8 negatively and Slamf1 positively affects macrophage migration upon peritonitis induction.
(A) Total number of peritoneal cells from wt, Slamf1-/- and Slamf8-/- Balb/c mice four days after i.p. injection of 4% thioglycollate. (B) Number of macrophages (CD11b+ F4/80+) among collected peritoneal cells. The data are representative of 3 independent experiments, each consisting of at least 3 mice per experimental condition.
Fig 3.
Slamf8 deficient mice show expedited macrophage repopulation of small intestine lamina propria after anti-CD3 mediated depletion.
Depletion of lamina propria macrophages 3 and 5 days after administration of αCD3. (A) The percentage of CD11b+ CD103- of total CD45.2+ CD11c+ Ly6G- lamina propria cells. (B) Representative dot plots (gated on CD45.2+ CD11c+ Ly6G-) showing macrophages in lamina propria isolated cells from wt (left) and Slamf8-/- (right) naïve mice (day 0) or injected with αCD3 3 days prior (middle), or 5 days prior (bottom). (C) Percentages of monocytes (MHC-IIlow/- Ly6C+) of total CD11b+ CD45.2+ that were isolated from the lamina propria on day 0, 3, or 5 after αCD3 administration. (D) Representative dot plots showing monocytes in the lamina propria of the small intestine of wt and Slamf8-/- mice on day 3. The data are representative of 2 independent experiments, each consisting of at least 3 mice per experimental condition.
Fig 4.
Slamf8-/- neutrophil produce higher ROS, and Slamf8 negatively regulates neutrophil migration.
(A) ROS production as measured by luminescence in wt and Slamf8-/- mice thio-neutrophils in response to heat inactivated E.coli F18 and PMA. (B) Total number of peritoneal cells obtained from wt and Slamf8-/- mice after 4 hours i.p. injection of 4% thioglycollate. (C) Number and (D) percentage of neutrophil (CD11b+ Ly6G+) among peritoneal cells in wt and Slamf8-/- mice. (E) Representative dot plots (gated on DAPI- cells) showing CD11b+ Ly6G+ neutrophil in the peritoneal cavity of wt and Slamf8-/- mice. (F) Slamf8-/- and wt mice were injected with αCD3. Percentage of neutrophils (CD11b+ Ly6Ghigh) from the small intestine lamina propria 0, 3 and 5 days after αCD3 injection. (G) Representative dot plots (gated on CD45.2+) showing neutrophils. The data are representative of 2 independent experiments, each consisting of at least 3 mice per experimental condition.
Fig 5.
Slamf8 negatively regulates in vitro migration of DCs and of macrophages, while Slamf1 is a positive regulator of the same process.
Wt, Slamf1-/-, and Slamf8-/- skin DCs were isolated and column-purified before they were allowed to migrate toward a concentration range of CCL21 [0–400nM]. The migration index of (A) Slamf8-/- vs. wt DCs and (B) wt vs. Slamf1-/- DCs are plotted. (C) Macrophages from wt mice were incubated with different concentrations of DPI (1μM and 5μM) for 15 minutes, and the Nox2 activity in macrophages was quantified upon PMA (1mg/mL) stimulation. The data are representative of two independent experiments. (D) Wt, Slamf1-/-, and Slamf8-/- thio-macrophages migration in the presence of CSF-1, with and without pre-incubation with DPI. All the data are representative of three independent experiments.
Fig 6.
Slamf8 is a homophilic receptor, and Slamf8-Fc enhances DCs migration in vivo.
Slamf8 receptor interacts in a homophilic manner. HEK 293 cells were transfected with Slamf8 (A) or Slamf1 (B), and the binding of Slamf1-Fc and Slamf8-Fc to these transfected cells was determined by flow cytometry. (C) Scheme for the Slamf8-Fc binding assay. The data are representative of three independent experiments. Slamf8-Fc enhances DCs migration in vivo. Wt Balb/c mice were i.p. injected with Slamf8-Fc or human IgG1. Three hours later, FITC painting assay was performed. (D) Percentages among CD11c+ cells (E) total number of migratory (MHC-IIhi FITC+) DCs in the draining LNs. (F) Representative dot plots (gated on CD11c+ cells) showing migratory MHC-IIhi FITC+ DCs in the draining lymph nodes. The data are representative of two independent experiments.
Fig 7.
Proposed model showing Slamf1 and Slamf8 are counterparts for the regulation of Nox2 generated ROS and cell migration.
In migratory phagocytes, the early actor Slamf1 enhances ROS-mediated migration signals; subsequently Slamf8 reduces ROS-mediated migration signals. Inflammatory signals enhance the activity of Slamf1, which induces Nox-2 mediated ROS production. Inflammatory mediators, such as IFN-γ subsequently increase Slamf8 expression and function. This leads to the suppression of ROS production resulting in the reduction in infiltration of phagocytes. Thus, Slamf1 and Slamf8 together, balance the extent of infiltration of inflammatory cells.