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FIGURE 3. Integrin ligand <t>MFG-E8</t> stimulates MerTK cleavage and phagocytosis, although MerTK ligands Gas6 and protein S have opposite and more subtle roles. A, representative immunoblots and corresponding quantification showing that amounts of sMerTK are increased when cells are incubated with POS and the integrin ligand MFG-E8 (MF) for 3 or 6 h as indicated, although Gas6 (G6) and protein S (PS) have a much more limited effect. Ligands dose, 2 g/ml. Mean S.D., n 3–4. B, in the same conditions as in A, relative levels of total POS phagocytosis, POS binding, and internalization (intern.) were similar to the sMerTK production profile, as indicated. Mean S.D. with no ligand control total phagocytosis (/) set as 1, n 4. The left x axis scale corresponds to both total POS phagocytosis and POS binding, and the right x axis scale to POS internalization. A and B, significance was assessed using one-way ANOVA comparisons versus either nontreated (A) or POS challenged cells (B); **, p 0.01; ***, p 0.001. C, all ligands similarly stimulated total phagocytic activity of J774 macrophages when various doses of ligands ranging from 1 to 10 g/ml were used either alone or in combination during 3 h of phagocytic challenge. D and E, in the same conditions, MFG-E8 greatly stimulated POS total phagocytosis (D) as well as internal (E) phagocytosis in RPE-J cells (RPE). Gas6 seems to bear an inhibitory role on POS phagocytosis as depicted on this phagocytosis quantification of RPE-J cells when protein S appeared stimulatory. Gas6 effect was dominant on total phagocytosis when used in combination with protein S, although MFG-E8 effect was superior to both MerTK ligands when all three were tested. Effects were more marked for POS internalization (E) compared with total phagocytosis (D, total binding internalization). C–E, mean S.D., POS without ligand (/) is set as 1, n 3 (D and E) to 5 (C). Significance was assessed using Student’s t test using POS alone (/) as reference; *, p 0.05; **, p 0.01; ***, p 0.001. ANOVA comparisons for dose-effect validation did not show any significant difference between the doses tested for each ligand series.
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FIGURE 3. Integrin ligand MFG-E8 stimulates MerTK cleavage and phagocytosis, although MerTK ligands Gas6 and protein S have opposite and more subtle roles. A, representative immunoblots and corresponding quantification showing that amounts of sMerTK are increased when cells are incubated with POS and the integrin ligand MFG-E8 (MF) for 3 or 6 h as indicated, although Gas6 (G6) and protein S (PS) have a much more limited effect. Ligands dose, 2 g/ml. Mean S.D., n 3–4. B, in the same conditions as in A, relative levels of total POS phagocytosis, POS binding, and internalization (intern.) were similar to the sMerTK production profile, as indicated. Mean S.D. with no ligand control total phagocytosis (/) set as 1, n 4. The left x axis scale corresponds to both total POS phagocytosis and POS binding, and the right x axis scale to POS internalization. A and B, significance was assessed using one-way ANOVA comparisons versus either nontreated (A) or POS challenged cells (B); **, p 0.01; ***, p 0.001. C, all ligands similarly stimulated total phagocytic activity of J774 macrophages when various doses of ligands ranging from 1 to 10 g/ml were used either alone or in combination during 3 h of phagocytic challenge. D and E, in the same conditions, MFG-E8 greatly stimulated POS total phagocytosis (D) as well as internal (E) phagocytosis in RPE-J cells (RPE). Gas6 seems to bear an inhibitory role on POS phagocytosis as depicted on this phagocytosis quantification of RPE-J cells when protein S appeared stimulatory. Gas6 effect was dominant on total phagocytosis when used in combination with protein S, although MFG-E8 effect was superior to both MerTK ligands when all three were tested. Effects were more marked for POS internalization (E) compared with total phagocytosis (D, total binding internalization). C–E, mean S.D., POS without ligand (/) is set as 1, n 3 (D and E) to 5 (C). Significance was assessed using Student’s t test using POS alone (/) as reference; *, p 0.05; **, p 0.01; ***, p 0.001. ANOVA comparisons for dose-effect validation did not show any significant difference between the doses tested for each ligand series.

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

doi: 10.1074/jbc.m114.628297

Figure Lengend Snippet: FIGURE 3. Integrin ligand MFG-E8 stimulates MerTK cleavage and phagocytosis, although MerTK ligands Gas6 and protein S have opposite and more subtle roles. A, representative immunoblots and corresponding quantification showing that amounts of sMerTK are increased when cells are incubated with POS and the integrin ligand MFG-E8 (MF) for 3 or 6 h as indicated, although Gas6 (G6) and protein S (PS) have a much more limited effect. Ligands dose, 2 g/ml. Mean S.D., n 3–4. B, in the same conditions as in A, relative levels of total POS phagocytosis, POS binding, and internalization (intern.) were similar to the sMerTK production profile, as indicated. Mean S.D. with no ligand control total phagocytosis (/) set as 1, n 4. The left x axis scale corresponds to both total POS phagocytosis and POS binding, and the right x axis scale to POS internalization. A and B, significance was assessed using one-way ANOVA comparisons versus either nontreated (A) or POS challenged cells (B); **, p 0.01; ***, p 0.001. C, all ligands similarly stimulated total phagocytic activity of J774 macrophages when various doses of ligands ranging from 1 to 10 g/ml were used either alone or in combination during 3 h of phagocytic challenge. D and E, in the same conditions, MFG-E8 greatly stimulated POS total phagocytosis (D) as well as internal (E) phagocytosis in RPE-J cells (RPE). Gas6 seems to bear an inhibitory role on POS phagocytosis as depicted on this phagocytosis quantification of RPE-J cells when protein S appeared stimulatory. Gas6 effect was dominant on total phagocytosis when used in combination with protein S, although MFG-E8 effect was superior to both MerTK ligands when all three were tested. Effects were more marked for POS internalization (E) compared with total phagocytosis (D, total binding internalization). C–E, mean S.D., POS without ligand (/) is set as 1, n 3 (D and E) to 5 (C). Significance was assessed using Student’s t test using POS alone (/) as reference; *, p 0.05; **, p 0.01; ***, p 0.001. ANOVA comparisons for dose-effect validation did not show any significant difference between the doses tested for each ligand series.

Article Snippet: Recombinant proteins and antibodies against mouse MerTK, mouse MFG-E8, and mouse Gas6 were from R & D Systems, and recombinant protein S was from MP Biomedicals.

Techniques: Western Blot, Incubation, Binding Assay, Control, Activity Assay, Biomarker Discovery

FIGURE 4. sMerTK acts as a decoy receptor. A, histogram representing the quantification of the relative total, binding, or internalization (intern.) phago- cyticcapacityofuntreatedRPE-Jcellswhenchallengedfor3hwithPOSresus- pended in regular medium (medium) or sMerTK-enriched CM concentrated from RPE-J (RPE CM) cells or J774 macrophages (M CM) incubated overnight with serum-free medium as indicated. Presence of sMerTK significantly impacts all steps of phagocytosis by RPE cells. Mean S.D., n 5, Student’s t test; *, p 0.05; **, p 0.01; ***, p 0.001. B, histogram representing the quantification of the relative total phagocytic capacity of untreated RPE-J cells when challenged for 3 h with POS resuspended in DMEM (med) with () or without ( ) rMerTK, in the presence of various ligands (2 g/ml) as indi- cated. rMerTK alone decreases phagocytosis, although it has no impact on the stimulatory effect of MFG-E8. rMerTK counteracts the inhibitory effect of Gas6 and the stimulatory effect of protein S. The lowest phagocytic levels were reached when rMerTK was present with both Gas6 and protein S. Mean S.D., n 4, Student’s t test, *, p 0.05; **, p 0.01.

Journal: Journal of Biological Chemistry

Article Title: Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

doi: 10.1074/jbc.m114.628297

Figure Lengend Snippet: FIGURE 4. sMerTK acts as a decoy receptor. A, histogram representing the quantification of the relative total, binding, or internalization (intern.) phago- cyticcapacityofuntreatedRPE-Jcellswhenchallengedfor3hwithPOSresus- pended in regular medium (medium) or sMerTK-enriched CM concentrated from RPE-J (RPE CM) cells or J774 macrophages (M CM) incubated overnight with serum-free medium as indicated. Presence of sMerTK significantly impacts all steps of phagocytosis by RPE cells. Mean S.D., n 5, Student’s t test; *, p 0.05; **, p 0.01; ***, p 0.001. B, histogram representing the quantification of the relative total phagocytic capacity of untreated RPE-J cells when challenged for 3 h with POS resuspended in DMEM (med) with () or without ( ) rMerTK, in the presence of various ligands (2 g/ml) as indi- cated. rMerTK alone decreases phagocytosis, although it has no impact on the stimulatory effect of MFG-E8. rMerTK counteracts the inhibitory effect of Gas6 and the stimulatory effect of protein S. The lowest phagocytic levels were reached when rMerTK was present with both Gas6 and protein S. Mean S.D., n 4, Student’s t test, *, p 0.05; **, p 0.01.

Article Snippet: Recombinant proteins and antibodies against mouse MerTK, mouse MFG-E8, and mouse Gas6 were from R & D Systems, and recombinant protein S was from MP Biomedicals.

Techniques: Binding Assay, Incubation