Figure 1.
BM disruption induces melanotic mass formation.
(A, B) Pictures (A) and percentages (B) of the third instar larvae of the different genotypes containing melanotic nodules. Hsp70-GAL4 was used. The control represents GAL4 only. n>150 for each genotype. Heat shock was carried out as described in Methods. (C–F) The larval fat bodies were analyzed for lamellocyte encapsulation. Hsp70>GAL4 was used. The control was GAL4 only. Anti-L1 (red) and phalloidin-FITC (green) marked F-actin-rich lamellocytes. Nuclei were stained with DAPI (blue). (G–J) Confocal images of the fat body BM. Hsp70-GAL4 was used. The control was GAL4-only. LanB2-i;+/− indicates Hsp70>LanB2-i;LanB2+/LanB2−. Collagen IV (Col IV), laminin, and nuclei were visualized after staining with anti-Col IV (green), anti-LanB1 (red), and DAPI (blue), respectively. (K) Quantitation of the fluorescence intensities for collagen IV in the BM after staining with anti-Col IV antibody (green) and for BM laminin after staining with anti-LanB1 (red). Error bars represent standard errors of the mean (SEM). *p<0.05, **p<0.01, and ***p<0.001 by Student's t test. For each genotype, n≥5. Scale bar: 500 µm (A), 100 µm (C–F), and 50 µm (G–J).
Figure 2.
Melanotic mass formation in the BM-deficient larvae is a normal immune response against altered self.
(A) Numbers of circulating hemocytes (blue+red) and lamellocytes (red) in each larva were counted in the various genotypes. Cg-GAL4 drives gene expression in hemocytes and the fat body. hopTum was raised at 29°C. Error bars represent SEM. ***p<0.001 by Student's t-test. Percentages of larvae containing melanotic masses: Cg>mys-i, 27.38% (n = 141/515); Cg>mys-i domeΔCYT, 32.00% (n = 72/225). (B) Lamellocyte differentiation in the lymph gland was analyzed by staining with anti-L1 antibodies (red) in the larvae of the indicated genotypes. The nuclei were stained with DAPI (blue). For the vkg and LanB2 knockdowns, two classes were observed, the frequency of which plus an example is shown. The control panel represents Oregon R. hopTum was raised at 25°C. Scale bar: 50 µm. (C) Melanotic mass formation after knockdown of genes for collagen IV, laminin, and integrin in various combinations using the fat body-specific FB-GAL4. For each genotype, n>150.
Figure 3.
Laminin alone sufficiently blocks melanotic mass formation.
(A–C) Confocal images of salivary-gland BMs after staining for collagen IV (anti-Col IV in green), laminin (anti-LanB1 in red), and nuclei (DAPI in blue). The control is GAL4-only. (D) Quantitation of the fluorescence intensities in (A–C). Error bars represent SEM. *p<0.05, ***p<0.001 by Student's t-test. (E–G) Salivary glands from (A–C) after staining lamellocytes using anti-L1 antibodies (red) and the nuclei with DAPI (blue). (H–J) Confocal images of salivary-gland BMs after visualizing collagen IV using Vkg-GFP (green), laminin using anti-LanB1 antibodies (red), and nuclei using DAPI (blue). HmlΔ-GAL4 and FB-GAL4 drive gene expression in hemocytes the fat body, respectively. The control represents vkgG454/+. (K) Quantitation of the fluorescence intensities in (H–J). Error bars represent SEM. **p<0.01 and ***p<0.001 by Student's t-test. (L) Percentages of larvae containing melanotic masses in (A–C) and (H–J) and their mass-forming positions. SG and FB indicate salivary gland and fat body, respectively. (M, N) Confocal images of the salivary-gland BMs of vkgG454/+ (M) and vkgG454 UAS-vkg-i/+ +; Hsp70-GAL4/UAS-trol-i (N) larvae after visualization of collagen IV using Vkg-GFP (green), Perlecan (Trol) using anti-Trol antibodies (purple), Nidogen using anti-Ndg antibodies (cyan), laminin using anti-LanB2 antibodies (red), and nuclei using DAPI (blue). Scale bar: 50 µm (A–C, H–J, M, N) and 200 µm (E–G).
Figure 4.
Cell integrity is severely disrupted specifically in BM-deficient, melanotic mass-containing larvae.
(A–J) Confocal images of larval salivary glands from the indicated genotypes illustrate defects in apicobasal cell polarity and cell-cell adhesion. Cora and Dlg were stained with anti-Cora (red; A, C, E, G, I) and anti-Dlg (red; B, D, F, H, J), respectively. F-actin and the nuclei were stained with phalloidin-FITC (green) and DAPI (blue), respectively. ptc-GAL4 is active in the salivary gland and various other tissues. AB1-GAL4 is active in the salivary gland. The control was Oregon R. Asterisks mark the lumen. (K) Confocal images of the salivary-gland BMs of AB1>mys-i larvae showing BM collagen IV (Vkg-GFP in green) and BM laminin (anti-LanB2 in red). (L) Confocal images of the salivary gland revealed that the organ surface is negative for the pan-hemocyte marker Hemese (Hem, red). Nuclei were stained with DAPI (blue). Scale bar: 50 µm (A–J, K) and 100 µm (L).
Figure 5.
Loss of cell integrity is required in addition to BM disruption for melanotic mass formation.
(A, B) Mechanical disruption (arrowhead) of the salivary-gland BM of AB1>mys-i, GFP larvae with GFP expression (green) in the salivary gland. (C) Melanotic mass formation in pinch-wounded larvae of AB1>GFP control (n = 1/57) and AB1>mys-i, GFP (n = 17/84) larvae. (D) Melanized salivary glands of wounded AB1>mys-i larvae were positive for L1 (red). Nuclei were stained with DAPI (blue). (E–J) Confocal images of the larval salivary glands of the indicated genotypes after staining lamellocytes with anti-L1 antibodies (red) and nuclei with DAPI (blue). Scale bar: 200 µm (A, B, E–G) and 100 µm (D, H–J).
Figure 6.
Proposed model for the self-tolerance checkpoint function in the Drosophila immune system.
Epithelial tissues are equipped with at least two self-tolerance checkpoints: BM laminin and cell integrity. The latter is currently less well-defined but appears to include apicobasal cell polarity and cell-cell adhesion. With either one of the checkpoints, self-tissue may become tolerant to the immune system of its own. Upon Mmp2 overexpression (OE), the salivary gland loses BM integrity to an extent that allows plasmatocyte access; however, cell integrity remains largely intact (intact 2nd checkpoint). Upon integrin knockdown (KD) in the salivary gland (AB1>mys-i), cells lose cell integrity, while the organ maintains the BM (intact 1st checkpoint). In the presence of laminin knockdown, both of the self-tolerance checkpoints are non-functional, and the tissues are subjected to lamellocyte encapsulation. Avirulent parasitoid eggs or wing disc implants from distantly related species [11] do not have either of the checkpoints that are compatible with the host immune system, and thus, these foreign bodies are also sequestered by lamellocyte encapsulation.