Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.

The proteolytic activity of caspases is involved in apoptosis and inflammation. In this regard, caspase-1 is required for pro-interleukin (IL)-1␤ and pro-IL-18 maturation. We report here on a novel function of caspase-1 as an activator of nuclear factor of the -enhancer in B-cells (NF-B) and p38 mitogen-activated protein kinase (MAPK). This function is not shared by the murine caspase-1 homologues caspase-11 and -12. In contrast to pro-IL-1␤ maturation, caspase-1-induced NF-B activation is not inhibited by the virus-derived caspase-1 inhibitor cytokine response modifier A and is equally induced by the enzymatically inactive caspase-1 C285A mutant. Although the general NF-B-inhibiting protein A20 inhibits caspase-1-derived activation of NF-B, dominant-negative forms of TRAF2 and RIP1 have no effect. We demonstrate that caspase-1 interacts with RIP2 and that dominant-negative forms of RIP2 and IB kinase complex-␤ inhibit caspase-1-mediated NF-B activation. Structure-function analysis shows that the caspase recruitment domain of caspase-1 mediates the activation of NF-B and p38 MAPK. These data demonstrate that caspase-1 contributes to inflammation by two distinct pathways: proteolysis of pro-IL-1␤, and RIP2-dependent activation of NF-B and p38 MAPK mediated by the caspase recruitment domain.

Phylogenetic analysis clusters caspase-12 with the inflammatory caspases 1 and 11. We analyzed the expression of caspase-12 in mouse embryos, adult organs, and different cell types and tested the effect of interferons (IFNs) and other proinflammatory stimuli. Constitutive expression of the caspase-12 protein was restricted to certain cell types, such as epithelial cells, primary fibroblasts, and L929 fibrosarcoma cells. In fibroblasts and B16/B16 melanoma cells, caspase-12 expression is stimulated by IFN-γ but not by IFN-α or -β. The effect is increased further when IFN-γ is combined with TNF, lipopolysaccharide (LPS), or dsRNA. These stimuli also induce caspase-1 and -11 but inhibit the expression of caspase-3 and -9. In contrast to caspase-1 and -11, no caspase-12 protein was detected in macrophages in any of these treatments. Transient overexpression of full-length caspase-12 leads to proteolytic processing of the enzyme and apoptosis. Similar processing occurs in TNF-, LPS-, Fas ligand–, and thapsigargin (Tg)-induced apoptosis. However, B16/B16 melanoma cells die when treated with the ER stress–inducing agent Tg whether they express caspase-12 or not.

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.