Resistance of cytolytic lymphocytes to perforin-mediated killing. Induction of resistance correlates with increase in cytotoxicity.

CTL and NK cells cultured in vitro are known to produce a cytolytic pore-forming protein (PFP, perforin) localized in their cytoplasmic granules. Using purified perforin, we showed here that both cloned CTL and primary killer cell populations, including allospecific CTL, NK/lymphokine-activated killer cells, and MHC-non-restricted CTL, were more resistant to perforin-mediated killing than other lymphocyte populations and cell types. Similar results were obtained with both murine and human cytolytic lymphocyte populations. Resistance of killer cells to perforin correlated in general with their cytolytic capability. Thus, cells that have acquired competence to kill after stimulation with Con A, IL-2, or leukocyte-conditioned medium, were also the more resistant cells. IL-2-independent CTL lines and hybridomas derived in our laboratories could be triggered to become cytotoxic and perforin resistant by short-term stimulation with various cytokines, indicating that the acquisition of resistance to perforin-mediated lysis was independent of cell proliferation. Activation of one IL-2-independent CTL line with IL-2 also resulted in enhanced production of perforin and in enhanced serine esterase activity. The acquisition of cell resistance to perforin by these IL-2-independent cell lines after activation with stimulatory reagents was independent of protein and RNA neosynthesis: emetine, cycloheximide, and actinomycin D, while effectively blocking the incorporation of [35S]methionine into cell proteins, did not affect the induced increase in perforin resistance.

Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model.

Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1beta-converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1beta and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1(-/)- mice) had an oral S. typhimurium 50% lethal dose (LD(50)) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1(-/)- mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1(-/)- mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1(-/)- mice after an oral dose of S. typhimurium that was 100-fold higher than the LD(50). The increased resistance in casp-1(-/)- animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.

Resistance of cytolytic lymphocytes to perforin-mediated killing. Lack of correlation with complement-associated homologous species restriction.

CTL and NK cells resist self-mediated killing and lysis by their own pore-forming protein (PFP; perforin). Perforin, like C, lyses RBC. Efficient C-mediated lysis of RBC occurs when both C and RBC are from different species (homologous species restriction). A protective surface protein (C8-binding protein, homologous restriction factor) has been reported to mediate both homologous species restriction in C-dependent cytolysis and protection of some target cells against perforin-induced lysis. We show here that perforin, unlike C, lyses target cells across a variety of species, including the homologous one, while the same target cell populations resist the attack by homologous C. Perforin-containing extracts of CTL and LAK/NK cells from three species (rat, mouse, and human) and purified mouse perforin were tested against RBC from 10 different species, several nucleated target cell lines, and one primary cell population (thymocytes). While resisting lysis by homologous C, most of these cell types were lysed effectively by perforin without any homologous restriction pattern. CTL and NK cells, like other nucleated targets, are resistant to lysis by homologous but not heterologous C; however, these cell types are resistant to both homologous and heterologous perforin. Together, our results suggest that the protective mechanisms associated with C- and perforin-mediated lysis are distinct.

Extracellular ATP as a trigger for apoptosis or programmed cell death.

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.

Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.

Interleukin 1 is released by murine macrophages during apoptosis induced by Shigella flexneri.

Peritoneal macrophages undergoing apoptosis induced by Shigella flexneri infection release the inflammatory cytokine interleukin 1 (IL-1), but not IL-6 or tumor necrosis factor alpha (TNF alpha). Wild type shigella causes a very fast and significant release of IL-1 from prestimulated peritoneal macrophages, before the cell's integrity is compromised. Both IL-1 alpha and IL-1 beta are released, IL-1 beta in its mature processed form. IL-1 is released from presynthesized cytoplasmic pools. These results demonstrate that bacteria-induced apoptosis of macrophages may play an active role in vivo by releasing IL-1, which in turn mediates an early inflammatory response in epithelial tissues.

How bacteria initiate inflammation: aspects of the emerging story.

Recent studies have shown that bacteria possess an array of proinflammatory molecules in addition to the extensively studied lipopolysaccharide and superantigens. These bacterial molecules include soluble and membrane-associated inducers of cytokine release, inducers of host cell apoptosis, and immunostimulatory DNA. There is therefore much greater diversity in the class of molecules and mechanisms by which bacteria engage the host immune system than previously appreciated.

Beyond the grave: When is cell death critical for immunity to infection?

Immune cell death is often observed in response to infection. There are three potential beneficial outcomes after host cell death: (1) the removal of an intracellular niche for microbes, (2) direct microbicidal activity of released components and (3) the propagation of an inflammatory response. Recent findings suggest that three forms of non-apoptotic regulated cell death, pyroptosis, necroptosis and NETosis, can impact on immunity to bacterial infection. However, it is challenging to design experiments that unequivocally prove the advantageous effects of regulated cell death on immunity. Recent advances in the genetic manipulation of regulated cell death and danger-associated molecular patterns and 'alarmins', such as HMGB1 and the IL-1 family, may hold the key to delineating the consequences of cell death in immunity to infection.

Programmed cell death in infectious diseases.

Apoptosis plays an important role during normal animal development, and in proliferative and degenerative diseases. It is perhaps not surprising that apoptosis can be manipulated by infectious microorganisms to their own advantage, either to promote host cell immortality or to combat cells of the immune system.

Apoptosis as a proinflammatory event: what can we learn from bacteria-induced cell death?

Infection of cells by some pathogenic bacteria triggers host cell apoptosis. Bacteria-induced apoptosis appears to promote an inflammatory response that causes tissue damage and further bacterial colonization. Shigella pathogenesis offers a paradigm for the role of apoptosis in bacterial infections.

The regulation of apoptosis by microbial pathogens.

In the past few years, there has been remarkable progress unraveling the mechanism and significance of eukaryotic programmed cell death (PCD), or apoptosis. Not surprisingly, it has been discovered that numerous, unrelated microbial pathogens engage or circumvent the host's apoptotic program. In this chapter, we briefly summarize apoptosis, emphasizing those studies which assist the reader in understanding the subsequent discussion on PCD and pathogens. We then examine the relationship between virulent bacteria and apoptosis. This section is organized to reflect both common and diverse mechanisms employed by bacteria to induce PCD. A short discussion of parasites and fungi is followed by a detailed description of the interaction of viral pathogens with the apoptotic machinery. Throughout the review, apoptosis is considered within the broader contexts of pathogenesis, virulence, and host defense. Our goals are to update the reader on this rapidly expanding field and identify topics in the current literature which demand further investigation.

Toll-like receptor-2 transduces signals for NF-kappa B activation, apoptosis and reactive oxygen species production.

The innate immune system coordinates the inflammatory response to pathogens. To do so, cells of the innate immune system must rapidly discriminate between self and non-self. All bacteria express membrane-associated lipoproteins. These molecules activate cells of the innate immune system to initiate host defense mechanisms. However, it is currently unknown how the innate immune system recognizes bacterial lipoproteins. Here, we describe that in response to bacterial lipoprotein, human Toll-like receptor-2 activates three different cellular responses: nuclear factor-kappaB dependent transcription, programmed cell death and reactive oxygen species production. We propose that Toll-like receptor-2 fulfils multiple roles in the genesis of the immune response to bacterial pathogens.

Molecular and cellular mechanisms of tissue invasion by Shigella flexneri.

Shigella flexneri, a member of the family of enterobacteriaceae, causes bacillary dysentery by invading the human colonic mucosa and provoking a very intense inflammation. Recent in vitro data allow us to integrate different phenomena into a model of the infectious process during shigellosis. In vivo, bacteria appear to enter the submucosa via the M cells, specialized cells that cover the follicular structures of the intestinal mucosa. Once inside the submucosa, shigellae encounter resident tissue macrophages, which are infected, and apoptosis is rapidly induced. During programmed cell death the inflammatory cytokine interleukin-1 (IL-1) is released. Interleukin-1 triggers an inflammatory reaction characterized by extravasation of polymorphonuclear (PMN) cells. The inflammation is probably potentiated by the production of other cytokines by epithelial, endothelial, and PMN cells. Polymorphonuclear cells migrate through the epithelium into the lumen of the colon, destabilizing the integrity of the epithelial barrier. The damaged epithelium allows massive entry of bacteria into the submucosa. Further colonization of the epithelium aggravates inflammation, which in turn causes extensive tissue destruction. Both the in vitro and in vivo results that support this model are discussed.

Spontaneous formation of IpaB ion channels in host cell membranes reveals how Shigella induces pyroptosis in macrophages.

The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria.

Sensing, presenting, and regulating PAMPs.

Recognition of microbial infection and initiation of immune responses are controlled by multiple mechanisms. Toll-like receptors (TLRs) are key components of the innate immune system that detect microbial infection. TLR activation helps to eliminate the invading pathogens, coordinate systemic defenses, and initiate adaptive immune responses. Despite progress elucidating the TLR signaling aspects and the physiological relevance of TLRs in microbial infections, the molecular basis of microbial recognition by TLRs is still not fully understood. In this article we focus on the availability of microbial ligands to regulate presentation to TLRs and assist in our understanding of TLR-mediated microbial recognition.

Apoptosis induced by bacterial pathogens.

Programmed cell death is part of normal development and homeostasis. Apoptosis induced by bacteria appears to contribute to infectious diseases. Some bacteria produce toxins to kill host cells by the same pathway, apoptosis, through different mechanisms including pore formation, protein synthesis inhibition or adenylate cyclase activity. Other bacterial pathogens' mechanisms to induce apoptosis, for example, that of S. flexneri, remain to be elucidated. How the bacterial toxins or the bacteria interact with eukaryotic cell-death-related genes and then possibly trigger a cell-death program would make an interesting study. The understanding of the mechanism of apoptosis induced by bacteria could be important in the development of therapy and prevention of infectious diseases.

Clinical isolates of Shigella species induce apoptosis in macrophages.

Shigella species are invasive enterobacteria that cause dysentery, a severe form of diarrhea. The ability to invade epithelial cells and to kill macrophages is essential for virulence in a prototype Shigella flexneri strain. It is shown here that clinical isolates of both S. flexneri and Shigella sonnei invade epithelial cells and are cytotoxic to macrophages in vitro. Furthermore, clinical Shigella strains kill macrophages by inducing apoptosis. The conservation of the ability to induce macrophage apoptosis by clinical isolates suggests that this function plays a crucial role in the pathogenesis of Shigella species.