Innate Immunity Part I: The Immediate Response Deborah A. Lebman, Ph.D.

OBJECTIVES

1. Describe three mechanisms present at epithelial surfaces that protect the host from pathogens. 2. List 3 functions of complement proteins in innate immunity 3. Know the primary functions of macrophages and neutrophils 4. List the receptors on macrophages involved in recognition of pathogens 5. List the key toxic products of phagocytes

READING

• Parham, The Immune System, pg 227-238.

TERMS

• Defensin: antibacterial peptide produced by the host at epithelial surfaces • Complement: ubiquitously present proteins synthesized primarily in the liver that mediate a variety of responses to pathogens • Opsonin (opsonization): alteration of cell surface that facilitates phagocytosis • Phagocytosis: internalization of particulate matter by cells • CD14: LPS receptor • TLR-4: toll like receptor 4

I. Introduction

A. The first response to pathogens occurs immediately B. The early response employs both mechanical and cell mediated mechanisms C. There is no cellular expansion, but there is cell recruitment D. A wide array of soluble mediators with both positive and negative effects, i.e. remove the pathogen but hurt the host, is released

II. Overview of Pathogens

A. There are four types of pathogen: bacteria, virus, fungus, parasite B. Table I: Examples of the Four Types of Common Human Pathogen Table 1: Examples of the Four Types of Common Human Pathogen

Table 1 (Continued): Examples of the Four Types of Common Human Pathogen

C. Pathogens can be intracellular or extracellular

Table 2: Examples of intracellular and extracellular pathogens. Notice that both innate and adaptive mechanisms are involved in the response to both types of pathogen.

D. Pathogens enter the host at either mucosal surfaces (internal epithelium) or through the skin

1. Mucosal surfaces include the airway, gastrointestinal and reproductive tract

2. Entry via the external epithelium requires an injury such as a wound or insect bite

3. In order to succeed, the pathogen must form an initial focus or colony

III. The First Defenses

A. Epithelial Surfaces

1. Both external and internal epithelia are “active” barriers

a. Tight junctions provide a mechanical barrier

b. The flow of both air and mucus prevents bacterial adherence 2. Several anti-bacterial products are secreted at epithelial surfaces

a. Enzymes: Lysozyme breaks bacterial cell walls

b. Defensins: antibacterial peptides produced by underlying cells

3. Normal flora defends against pathogenic flora

a. Flora can be both active and passive

b. “passive” –occupies space preventing pathogens from gaining a foothold

c. “active”—secrete anti-microbial substances, e.g., colicins

d. Clinical note: a common problem following treatment with antibiotics is candidiasis. When the antibiotic kills normal flora along with pathogenic organism, it becomes possible for yeast to colonize epithelium.

Table 3: The three levels of protection provided by epithelial barriers.

B. Complement

1. Complement consists of over 30 plasma proteins. >90% are produced in the liver. Some of the proteins are produced by moncytes/macrophages and fibroblasts

2. Complement proteins are always present and ready which means that as soon as the exterior boundaries are breached, complement comes into play.

3. There are multiple pathways for activation of complement (another lecture)

4. The pathway of complement activation at this early time point in an infection is the alternative pathway.

5. Key steps in the alternative pathway of activation

6. C3 undergoes spontaneous hydrolysis (this occurs all the time)

a. Hydrolyzed C3 interacts with factor B and is cleaved to C3b and C3a

b. C3b “dies” unless it binds to a cell

c. On the cell surface, C3b interacts with factor B which is cleaved to Bb by factor D

d. Stabilized C3bBb is an active complex that “converts” or cleaves C3 into C3b and allows C3b to coat the cell surface

i. On bacterial surfaces properdin stabilizes C3bBb

e. C3b is the functional moiety:

i. It is a ligand for a macrophage receptor so it promotes phagocytosis (C3b is an opsonin) ii. It also promotes formation of a complex of complement proteins (membrane attack complex) that punches holes in cell surfaces

Figure 1: Steps in the formation of the active complex C3bBb

iii. Host cells are spared because C3bBb is not formed or stabilized on the host cell surface iv. Host cells express several proteins (CR1, DAF, MCP, Factor H) that disrupts C3bBb

Figure 2: Complement is activated on pathogen, but not host cells because host cells make proteins that prevent formation of C3bBb on the surface and deposition of C3b.

v. A third function of complement is to promote inflammation vi. C3b production also leads to the activation of other components of complement, most notably C5a,C3a, and C4a, which act on blood vessels to increase permeability ultimately leading to cellular influx and inflammation Figure 3: Complement components promote vascular leakage and inflammatory response.

IV. The Immune Cell Mediated Early Innate Response

A. Effector Cells

1. Macrophages/Monocytes

a. Macrophages which are derived from blood monocytes reside in tissue. They tend to be localized to connective tissue, the aerodigestive tract, the genitourinary tract and the liver. Liver macrophages are Kupfer cells. Macrophages are long lived.

b. Macrophages have three primary functions: phagocytosis, secrection of enzymes (very important, this leads to the induced innate immune response), and antigen presentation. They are a key player in both innate and adaptive immunity.

2. Neutrophils a.k.a. Polymorphonuclear leukoctyes (PMN) a. Smaller than macrophages

b. Granulocytes

c. Most abundant white blood cell

d. Not present in healthy tissue

e. Short-lived

f. Primary functions of PMNs are phagocytosis and cytokine production

3. NK cells a.k.a. Large Granular Lymphocytes

a. Major role is in viral infection

b. Primary functions of NK cells are killing of virally infected cells and cytokine production

4. Miscellaneous cell types

a. There are two types of cell that are believed to interface the innate and adaptive immune responses: T cells and B-1 B cells

B. Recognition of Pathogens

1. Danger vs Stranger

a. Innate immune cells have to distinguish self from non-self as well as non- pathogenic from pathogenic micro-organisms

b. Two hypotheses as to how “recognition” is accomplished.

i. The Danger Hypothesis: pathogens alert the host by causing tissue damage, i.e, a Danger Signal ii. The Stranger Hypothesis: pathogens have molecular patterns that are recognized by receptors on host immune cells

2. Danger Signals

a. Until recently, there was very little convincing evidence for the existence of danger signals. There were a couple of intriguing possibilities such as heat shock proteins.

b. Last Fall (October 2003), Shi, et al reported in Nature the existence of a “true” danger signal. Invading micro-organisms stimulate epithelial cells to release uric acid crystals which activate dendritic cells (potent antigen presenting cells).

c. Clinical note: Gout, an acute form of arthritis associated with inflammation, is caused by uric acid crystals in joints. However, the relevance of this association is at the moment unclear.

3. Stranger Signals and The Receptors on Immune Cells

a. Macrophages and neutrophils have receptors for components of complement (CR3, CR4)

i. Complement coating of pathogens targets them for opsonization via these receptors

b. Pattern Recognition Molecules

i. Microbe surfaces have repeating structural patterns or PAMP (pathogen associated molecular patterns) ii. Immune cells have receptors for PAMPs

• Mannose receptor, glycan receptor, and scavenger receptor recognize carbohydrates • CD14 or the LPS receptor recognizes lipopolysaccharide which is a component of the cell walll of gram negative bacteria, e.g., E. coli

Figure 4: Receptors on phagocytes lead to recognition and engulfment of bacteria.

4. Toll Receptors

a. Toll receptors are a family of proteins (10 to date) that contribute to signal transduction induced by the interaction of PAMPs with their receptors. They do not recognize PAMPs.

b. May respond to damage signals induced by inflammation

c. Clinical note: Toll receptors or mutations in toll receptors appear to play a role in inflammatory diseases including Crohn’s disease and atherosclerosis. This is a very young field, but therapeutic interventions that involve manipulations of these receptors are on the horizon.

d. TLR-4

i. Responsible for signal transduction following binding to CD14 ii. Signaling results in induction of expression of surface proteins that enhance adaptive immunity and cytokine production

Figure 5: Following ligation of CD14 with bacterial LPS, TLR-4 induces cell signaling resulting in initiation of transcription of genes for both cell surface proteins and cytokines. A key transcription factor in this process is NF- B C. The initial cellular response: Phagocytosis

1. Recognition leads to engulfment of phagocytosis by macrophages and neutrophils

2. Endocytosed or phagocytosed microbe forms a membrane bound vesicle called a phagosome

3. Phagosome fuses with a lysozome to form a phagolysozome. The pH drops which can kill the micro-organism.

4. Phagocytes (macrophages and neutrophils) produce a variety of toxic products

a. The respiratory burst

i. Lysozomes have NADPH oxidases which generate toxic oxygen species including hydrogen peroxide, superoxide anions and nitric oxide. This early response is called the respiratory burst. ii. Clinical note: There are individuals who have defects in NADPH oxidase. They are susceptible to bacterial infections

b. Antimicrobial peptides

i. Defensins, cationic proteins

c. Enzymes

i. Myeloperoxidase: generates toxic oxygen derivatives ii. Lysozyme: dissolves gram negative cell walls iii. Acid hydrolases: digest bacteria

d. Competitors

i. Substances that compete with the bacteria for nutrients, e.g., lactoferrin which binds iron and Vitamin B12 binding protein