InflammationINTRODUCTION

BoneMarrowMyelopoiesis

Secondary Lymph nodes etc.

Apoptosis / Autophagy

CSF a.o.Haematopoietins

Margination & Diapedesis

Pathogen !Inflammation &Immunity

Resident WBC

PATHOGENS:

EXTRACELLULAR;

(E.G. E.COLI, S. TYPHI, A.O.)

INTRA-CELLULAR;

•CYTOSOLIC (E.G. LYSTERIA SP.)

•VACUOLAR (E.G. MYCOBACTERIA SP.)

OUTOME OF INFECTION:

PATHOGEN VIRULENCE FACTORS

Or / both

HOST CHARACTERISTICS

I. Introduction -Nonspecific defenses are characterized by immediate action and the inability to learn from experience; i.e., they do not respond any better or more quickly the second or tenth time they are exposed to a pathogen than they did the first time. II. Barriers - Barriers not only help maintain the internal milieu but also prevent the entry of most pathogens into the body. III. Competition - Competition for nutrients and susceptibility to antimicrobial factors released by other microorganisms restrict the types of bacteria which can live on skin and in the gastrointestinal tract. IV. Physiological responses - The host may respond to an infection with fever, which usually slows the growth rate of the pathogens. Infections usually induce inflammation which hinders the spread of the pathogen into general circulation and which delivers soluble and cellular resistance factors to the affected area. A. Inflammatory response B. Fever V. Soluble factors - Soluble protective factors include lytic enzymes and components of the acute phase response. VI. Phagocytic System - A. Introduction - The main function of some cells is phagocytosis. They are often called professional phagocytes. B. Phagocytic cells - Polymorphonuclear neutrophils (PMNs) are the most numerous of the white blood cells. They are the first phagocytic cells to arrive in large numbers and are responsible for ingesting and killing most of the bacteria at the site of an infection. The monocyte-macrophage lineage cells take 12 to 24 hours to collect in significant numbers at the site of an infection. They are more potent killers than the PMNs and tend to mop-up the pathogens that the PMNs were unable to handle. C. Steps Involved in Phagocytosis - Destruction of pathogens by phagocytes depends on successful completion of several activities. D. Granulocytes - Other granulocytes also serve as important effectors of immune responses. VII. Cytotoxic Mechanisms - Phagocytes have several cytotoxic mechanisms that work together to kill potential pathogens. VIII. Summary

Introduction The complete immune response with organized cellular and soluble components does not appear until the level of chordates. This means that organisms below that level are not able to develop immunity to diseases. However, since there are more non-chordates than chordates, they obviously have mechanisms to resist invasion by potential pathogens. This chapter will deal with those defense mechanisms which are collectively called nonspecific defenses. Chordates have retained all those nonspecific defense mechanisms and have added the immune system on top of the other systems. Indeed, many of the products of immune responses act to make the preexisting nonspecific defenses more effective. Nonspecific defenses are characterized by the ability to respond to a potential invader immediately (no lag period), and they act on all the invaders in the area. (There are seldom situations in which only a single potential pathogen gains access to the body.) Repeated exposure to the pathogen does not increase the speed with which the nonspecific defenses act nor their level of activity; i.e., no memory develops. Nonspecific host defenses may be grouped into five classes: mechanical barriers, competition, physiological responses such as fever and inflammation, soluble factors, and phagocytic cells.

Barriers Barriers are found at all surfaces exposed to the external environment. The most obvious of these is the skin. As long as it is intact, few organisms are able to penetrate to the interior of the body to establish an infection. The skin also secretes fatty acids, thereby preventing the growth of many types of bacteria (those that cannot live in an acid environment). The gastrointestinal, respiratory, and (to a lesser extent) the genito-urinary tracts are exposed to the external environment. These areas are lined with mucous membranes; i.e., cuboidal epithelium that secretes mucous. The mucous is continuously removed (along with any trapped microorganisms or particles) by the wave action of the cilia which covers the mucosal surface of these cells. Even the eyeball has a tough epithelial layer that is fairly resistant to penetration. Of course, it is also continually washed by tears.

Competition Only about 300 of the vast number of microorganisms are pathogenic in normal people. Some of the normally nonpathogenic bacteria live in the gastrointestinal tract. Others live on the skin or hair, in the hair follicles and the openings to the sebaceous gland, etc. These colonies are well established and adapted to their environments. When potential pathogens try to establish themselves in one of these environments as a prelude to gaining access to the body, they must compete with prior residents for nutrients and (in the GI tract) for oxygen. In addition, many of the comensal bacteria secrete factors that inhibit the growth of some other bacteria or fungi. Thus, many pathogens are not able to take the first step required to cause an infection.

Physiological ResponsesInflammatory ResponseThe inflammatory response is a series of nonspecific reactions to tissue injury and/or infection that is designed to bring phagocytic cells and large molecular weight molecules to the area and to somewhat isolate the area from the rest of the body by reducing venous and lymphatic return. The initial response is vasodilation that produces the typical warmth, redness, and swelling (due to fluid exudate through the gaps between endothelial cells produced as a result of the vasodilation). There are several factors that singly or together induce the inflammation. Tissue injury activates the kinin (or contact activation) system resulting in production of bradykinin which increases vascular permeability, decreases arterial resistance and causes smooth muscle contraction. (It is also responsible for most of the pain associated with the inflammatory response.) Fragments of complement fixation (C3a and C5a) bind to specific receptors on mast cells which signals them to degranulate. The granules contain histamine, leukotrienes and heparin which contribute strongly to the inflammatory response. Escape of the fluid through the gaps between the endothelial cells increases the cell to fluid ratio in the capillaries causing slowing of the blood with a corresponding slowing of blood flow through the venules. This facilitates margination of leukocytes (mostly neutrophils) and allows them to bind to receptors on endothelial cells induced by the inflammatory response. Interaction of leukocytes with endothelial cells is normally relatively low. However, factors (TNF, IL-1, IL-8) produced during an inflammatory response and/or endotoxin (lipopolysaccharide, LPS) from gram negative cell walls induces endothelial cells to express P-selectin which are then bound by L-selectin on leukocytes. This further slows movement of leukocytes along the vessels (generally called rolling). Activation of endothelial cells by inflammatory mediators causes them to express PAF (platelet activating factor) and to upregulate ICAM-1 (intercellular adhesion molecule) which are bound by G-proteins and integrins respectively on PMNs. These additional interactions firmly anchor PMNs to the vessel walls. They then migrate (diapedisis) through the gaps in response to chemotactic factors produced during the inflammatory response.

PAF has the additional effect of aggregating platelets at the site of inflammation, a process which is exacerbated by some of the prostaglandins. The platelets release serotonin which further increases vascular permeability. Thrombi initiated by the aggregated platelets may block blood flow to the extent that ischemic necrosis may result. The major effect of inflammation is to get elements of soluble and cellular (particularly polymorphonuclear leukocytes) host defenses into the area of injury. Although inflammation is painful and may cause tissue injury (especially if it is intense), it is a necessary response to injury. Fever Pathogens, like all other living organisms have an optimum temperature range for growth. Fever may raise the temperature above that optimum for a given organism thereby slowing its growth. The reduced growth rate may allow the body's defenses to more easily eradicate the pathogen. The elevated temperature may also speed up some of the body's defense mechanisms.

.

Soluble Factors Blood, hemolymph, and secretions contain various factors which inactivate or kill many pathogens before they can establish a clinically apparent infection. These include the acid environment of the stomach and (to a lesser extent) skin, enzymes such as lysozyme which attacks the cell walls of some types of bacteria, and DNAse and RNAse that destroy any unprotected RNA or DNA they encounter. Tissue injury induces production of several enzymes and factors collectively called acute phase proteins. One of the earliest to be discovered was C-reactive protein or CRP. It binds to some species of streptococci and activates complement. It also opsonizes them for removal by PMNs. Other acute phase proteins with important protective activity include 1-antichymotrypsin and the 1-antiproteinases. They inhibit the activity of several bacterial proteinases thereby inhibiting the spread of these bacteria from the site of the original infection

Reticuloendothelial system

Introduction Virtually all multicellular animals have motile cells that wander around and engulf (phagocytize) things. By means that are not yet well understood, they are able to distinguish between normal host tissue and potential pathogens or damaged tissue. It is known that pathogens like to eat things that are negatively charged which includes most bacteria and damaged tissue. Although this process is not very efficient, it is probably the most important (except for barriers) protective mechanism available to nonchordates. Indeed, if something goes wrong with this system in chordates, they will not survive long even with a perfectly functional immune system. Many products of immune responses are designed to stimulate phagocytes to greater and more efficient activity.

Phagocytic Cells General Ultimately, most pathogens are killed by phagocytes. Most cells have some phagocytic ability. The two most important cell types whose major function is phagocytosis are polymorphonuclear leukocytes (PMNs) and the monocyte-macrophage lineage cells (monocytes, macrophages (M), Kupffer cells, Langerhans cells, dendritic cells, and glial cells). They are sometimes known as professional phagocytes. Phagocytes face two major problems: they must come into contact with the potential pathogen and they must recognize the pathogen as something that must be destroyed.

M PMN

CR 1, 2, & 3 (receptors that bind with different affinity to C3b, C3bi, and C4b)

X X

FcR I (receptor for IgG 1 & 3) X

MHC class I (receptor for CD8) X X

MHC class II (receptor for CD4) X

Chemotaxis: The first problem is solved by chemotactic factors. Phagocytes have receptors scattered uniformly over their surfaces. The phagocytes move in the direction of the part of the surface that has the most receptors filled; i.e., toward the higher concentration of factor. The most important chemotactic factors are peptides that start with formyl-methionine (which are made by bacteria but not animals), C5a, and C3a (about 1% of the activity of C5a).

Opsonization: Phagocytes have some intrinsic ability to recognize many bacteria and damaged tissue as needing to be removed. However, this process is significantly enhanced by factors that label foreign material for the phagocytes (opsonins). One requirement for a substance to serve as an opsonin is for the phagocyte to have a membrane receptor that binds to the opsonin. As discussed earlier in the chapter, C3b is an excellent opsonin because PMNs and macrophages have receptors for C3b (and some of its breakdown products). The other major opsonin is immunoglobulin G.

? Dendritic Cells

PHAGOCYTES LEUKOCYTES WITH THE CAPACITY TO ENGULF PARTICULATE MATTER INCLUDING PATHOGENIC MICRO‑ORGANISMS: POLYMORPHONUCLEAR LEUKOCYTES / GRANULOCYTESMONONUCLEAR PHAGOCYTES / MACROPHAGES < MONOCYTES GRANULOCYTES = POLYMORPHONUCLEAR LEUKOCYTES WITH GRANULES IN CYTOPLASM. PRECURSORS = PROMYELOCYTES HAVE PRIMARY AZUROPHILIC GRANULES WHICH CONTAIN PEROXIDASE, ACIDIC HYDROLASE, NEUTRAL PROTEASES, PHOSPHOLIPASE, BACTERICIDAL PROTEIN AND LYSOZYME. MYELOPEROXIDASE IS A SPECIFIC BIOCHEMICAL MARKER OF GRANULOCYTES AND MONOCYTES MYELOCYTES AND MORE DIFFERENTIATED CELLS HAVE SECONDARY GRANULES WHICH GIVE THE SPECIFIC REACTIONS OFNEUTROPHILS; LYSOZYME, LACTOFERRIN, H2O2, & 0 .EOSINOPHILS; HISTAMINE, ARYL SULPHATASE, SRS‑ABASOPHILS; HEPARIN, HISTAMINE, SRS‑A & R‑ Fc IgE LEUKOCYTE ALKALINE PHOSPHATASE (LAP) DISTINGUISHES BETWEEN THE LEUKOCYTOSIS OF LEUKAEMIA (DECREASED) FROM THAT OF INFECTION / INFLAMMATION (INCREASED). MARGINATE IN CIRCULATION AND RESPOND TO CHEMOKINES / CHEMOCYTOKINES BY EMIGRATION INTO INTERSTITIAL SPACES. REACTIONS INVOLVE CELL‑CELL INTERACTIONS WITH ENDOTHELIUM AND CELL ADHESION MOLECULES (SELECTINS). PARTICIPATE IN HOST DEFENSE MECHANISMS BY1. RELEASE OF GRANULAR CONTENTS2. RELEASE OF CYTOKINES / CSF3. PHAGOCYTIC OXYGEN DEPENDENT KILLING

Phagocytic Cells Polymorphonuclear neutrophils The figure at the right is a light micrograph of polymorphonuclear neutrophils (PMNs), the most common type of white blood cell. They comprise about 50% - 70% of the leukocytes in circulation. At any given time, about half of the PMNs in the vascular system are marginated (i.e., adhering to the endothelial cells of the vessel walls). They range from 12 to 20 m in diameter.

Polymorphonuclear neutrophils (H & E stain, courtesy of J. Krause, Dept. of Pathology, Medical

College of Georgia

PMNs are named for their multilobed nucleus. (The DNA is continuous, as can be seen in serial sections of a PMN. It only looks like the lobes are independent when one examines a single slice.) PMNs belong to the class of leukocytes called granulocytes because their cytoplasm is filled with granules. (Technically the other granulocytes, basophils/mast cells and eosinophils are PMNs in that they also have multilobed nuclei. However, in practice, the abbreviation PMN refers to neutrophils only.) The granulocytes of a PMN are at a neutral pH, so they are also known as neutrophils. PMNs live about 1-2 days, so they must be continuously replaced by the bone marrow. They cannot replace their lysosomal enzymes, so after they engulf and kill several bacteria, they too die. PMNs are the "marines" of phagocytic cells. They are usually the first to arrive on the scene in any numbers and try to get the situation under control. Given their numbers and early arrival, they usually kill more of the bacterial invaders than other cell types. However, they are not very effective against eukaryotic pathogens (fungi and parasites).

Copyright ©2009 American Society of Hematology. Copyright restrictions may apply.

Delvaeye, M. et al. Blood 2009;114:2367-2374

Figure 1 Interplay between coagulation and innate immune pathways in response to DAMPs