Thursday, November 24, 2011

Cellular and Humoral Response Involved in Gram-Negative Bacterial Sepsis and Septic Shock

As soon as a bacterium enters the body, it is confronted with two lines of defense: a humoral line and a cellular line. The humoral factors comprise complement, antibodies, and acute phase proteins. In the cellular line of defense, in particular the mononuclear cells (monocytes and macrophages) and the neutrophils are of great significance since these cells may recognize bacterial cell wall constituents directly or indirectly after complement and antibody bind to the bacterium and its constituents. It is now thought that continuous challenges with small amounts of bacterial constituents may be necessary to keep the immune system alert to infections. Indeed, low levels of LPS are present in healthy individuals without causing disease (Takakuwa, et al., 1994; Vogel, et al., 1990).


While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a

prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).

The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the

terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:

LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).

In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,

IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).

The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).

The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as

IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).

Humeral response

Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate

these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)

During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).

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