Color plates

Plate 1. Decreased clearance of apoptotic thymocytes in mer-deficient mice. (A) The

histologic appearance of the thymus of a mer KO mouse given 2.5 mg of cortisone acetate

48 hours before sacrifice. Note the large numbers of darkly staining apoptotic cells in

the cortex, which reflect defective clearance. (B) The thymus of a wild-type (C57BL/6J)

mouse treated similarly. There are rare apoptotic cells in the thymus, indicating that

the monocyte-macrophage system has successfully engulfed them and carried them away

from the thymus (the authors’ unpublished data, 1999). (See also Fig. 1 in article by Cohen

and Caricchio.)

Plate 2. Accessibility of SSA/Ro proteins on apoptotic cardiocytes to maternal auto-

antibodies. For all fields shown, cultured human fetal cardiocytes were incubated with

anti-human Fas or isotype (7 hours, 37�C). Cells were not permeabilized before staining.

Cells were incubated with affinity purified anti-SSA/Ro/anti-SSB/La or normal human

IgG. The media was removed and the cells were washed twice in phosphate buffered saline

with calcium. Cells were fixed with 4% paraformaldehyde (20 min, 22�C), then incubated

with TRITC anti-IgG (red) and Hoechst (blue). (See also Fig. 2 in article by Clancy

and Buyon.)

Plate 3. Evaluation of apoptosis in histologic sections of fetal/neonatal hearts. (A, B)

Longitudinal section through the septum of a heart from a 22-week fetus who had CHB.

Apoptotic cells were identified by TUNEL FITC staining (A) and TUNEL peroxidase (B).

Original magnification �40. (C) Values are the mean apoptotic index. One hundred cells

were counted in three to five fields for each cardiac section. (See also Fig. 3 in article by

Clancy and Buyon.)

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Plate 1

Plate 3

Plate 4. Histologic evidence of increased levels of Fas and FADD in conduction tissue

from the heart of a 22-week fetus with CHB, but not in conduction tissue from the heart

of a normal 23-week fetus. Longitudinal sections through septa of a 22-week fetal heart

with CHB (A, C) and of a 23-week normal fetal heart (B, D) were stained with primary

(anti-Fas [A, B]; anti-FADD [C, D]) and secondary (species-specific alkaline phosphatase-

conjugated anti-IgG [A–D]). (See also Fig. 4 in article by Clancy and Buyon.)

Plate 5. (A) Hematoxylin-eosin staining of osteoarthritic cartilage shows a typical

chondrocyte lacuna that does not seem to contain a viable cell (arrowhead). (B,C)

Demonstration of apoptotic cells by the TUNEL technique in osteoarthritic articular car-

tilage. (D) Confocal laser-scanning microscopy of osteoarthritic (D) articular chondrocytes

using triple labeling techniques shows viable cells as scanned in Fig. 2 (E) Evaluation of

cell viability by demonstration of 18S rRNA in one osteoarthritic cartilage sample shows

multiple cell clusters in the upper zones. Original magnifications: (A)�200; (B,C,E)�100;

(D) �500. (See also Fig. 1 in article by Aigner et al.)

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Plate 5

Plate 6. Z-series of laser-scanning confocal microscopy analysis shows one of the

definitively empty lacunae (arrowheads) lying next to a filled lacuna in a specimen of

osteoarthritic articular cartilage (green: immunostaining for vimentin and unspecific

autofluorescence of the cartilage matrix; red: staining with propidium iodide for nuclear

DNA). A thick cartilage slice (70–100 mm) was scanned in 10-mm steps showing two

adjacent closed (intact) chondrocyte lacunae (original magnification, �200). (Reproduced

from Aigner T, Hemmel M, Neureiter D, Gebhard PM, Zeiler G, Kirchner T, et al.

Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritic

human articular knee cartilage: a study of proliferation, programmed cell death [apoptosis],

and viability of chondrocytes in normal and osteoarthritic human knee cartilage. Arthritis

Rheum 2001;44:1304-12; with permission.) (See also Fig. 2 in article by Aigner et al.)

Plate 7. Photomicrograph of a renal biopsy sample from a patient with necrotizing

glomerulonephritis. The arrow indicates an apoptotic cell with condensed nuclear

chromatin. Note the halo that surrounds the apoptotic cell, indicating that the cell lies

within a phagolysosome of a phagocyte (Haematoxylin and eosin stain, original mag-

nification �400). (Courtesy of C. Bellamy, MD, PhD, Edinburgh, United Kingdom.)

(See also Fig. 1 in article by Hughes et al.)

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Plate 7

Plate 8. Light microscopy of 1-mm tissue section of renal biopsy sample of immune

complex glomerulonephritis in the rat (24-hour time point, �1000). An apoptotic cell

(arrow) exhibiting marked chromatin condensation is evident within the lumen of a

glomerular capillary. (From Hughes J, Johnson RJ, Mooney A, Hugo C, Gordon K, Savill

J. Neutrophil fate in experimental glomerular capillary injury in the rat. Emigration ex-

ceeds in situ clearance by apoptosis. Am J Pathol 1997;150:223–34; with permission.)

(See also Fig. 2A in article by Hughes et al.)

Plate 9. TUNEL staining of renal tissue demonstrates an apoptotic interstitial cell. The

arrow indicates a cell that exhibits both Fx1A staining and a TUNEL positive nucleus

(original magnification �1000). (See also Fig. 2B in article by Hughes et al.)

Plate 10. Double labeling of renal tissue using TUNEL staining and immunostaining with

the Fx1A antibody that specifically stains the proximal tubular brush border demonstrates

an apoptotic proximal tubular cell (arrow; original magnification �1000). (See also Fig.

2C in article by Hughes et al.)

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Plate 9

Plate 10

Plate 11. Cytospin preparation of human mesangial cells cultured in full RPMI 1640

medium with 10% serum (original magnification �250). Note the normal nuclear

morphology with an open pattern of chromatin. (See also Fig. 3A in article by Hughes et al.)

Plate 12. Cytospin preparation of human mesangial cells (original magnification �250)

following prolonged serum deprivation. Many of the cells exhibit typical light microscopic

features of apoptosis (arrow) with condensed chromatin and cytoplasm. (See also Fig. 3B

in article by Hughes et al.)

Plate 11

Plate 12

Plate 13. Mesangial cells are able to phagocytose apoptotic cells. Light microscopy of

cultured human mesangial cells following a 3-hour interaction with apoptotic human

neutrophils. Following stringent washing, the monolayer is fixed and stained to detect the

presence of the neutrophil enzyme myeloperoxidase, which is not present in mesangial

cells. Spindle-shaped mesangial cells are seen containing numerous ingested brown neu-

trophils (original magnification �1000). (See also Fig. 4A in article by Hughes et al.)

Plate 14. Mesangial cells are able to phagocytose apoptotic cells. Cytospin preparation of

trypsinized human mesangial cells following a 3-hour interaction with apoptotic

neutrophils demonstrates mesangial cell ingestion of neutrophils (original magnification

�1000). (See also Fig. 4B in article by Hughes et al.)

Plate 15. Mesangial cells are able to phagocytose apoptotic cells. Cytospin preparation of

trypsinized human mesangial cells following a 3-hour interaction with apoptotic mesangial

cells demonstrates that mesangial cells are capable of phagocytosing apoptotic mesangial

cells (original magnification �1000). (See also Fig. 4C in article by Hughes et al.)

Plate 13

Plate 15

Plate 14

Plate 16. RP3 immunostaining demonstrates neutrophil infiltration in immune complex

glomerulonephritis (4-h time point, original magnification �1000). (From Hughes J,

Johnson RJ, Mooney A, Hugo C, Gordon K, Savill J. Neutrophil fate in experimental glo-

merular capillary injury in the rat. Emigration exceeds in situ clearance by apoptosis. Am J

Pathol 1997;150:223–34; with permission.) (See also Fig. 5A in article by Hughes et al.)

Plate 17. Autoradiography demonstrates the presence of a radiolabeled neutrophil (arrow)

within the glomerulus following the administration of 111Indium radiolabeled neutrophils

to rats before the initiation of glomerular inflammation (original magnification �800).

(From Hughes J, Johnson RJ, Mooney A, Hugo C, Gordon K, Savill J. Neutrophil fate in

experimental glomerular capillary injury in the rat. Emigration exceeds in situ clearance by

apoptosis. Am J Pathol 1997;150:223–34; with permission.) (See also Fig. 5B in article by

Hughes et al.)

Plate 18. Autoradiograph of renal tissue (24-hour biopsy specimen) immunostained with

the monoclonal antibody ED1 specific for the rat macrophage demonstrates a discrete

focus of autoradiographic grains localized to a glomerular ED1-positive cell (arrow)

suggesting clearance of apoptotic neutrophils by glomerular macrophages (original mag-

nification �1000). (From Hughes J, Johnson RJ, Mooney A, Hugo C, Gordon K, Savill J.

Neutrophil fate in experimental glomerular capillary injury in the rat. Emigration exceeds

in situ clearance by apoptosis. Am J Pathol 1997;150:223–34; with permission.) (See also

Fig. 5C in article by Hughes et al.)

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Plate 17

Plate 18