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Innate Immunity:
Off-the-Shelf Responses to Pathogens

This module will help you

• learn how antigen stimulates an innate immune response.
• see how leukocytes get into the infection site.
• learn how pathogen is eliminated.
• test your knowledge and immunology problem-solving skills.

Infection and Immunity Part 1
Migration and Adhesion
Pathogen Elimination
Key Concepts

Infection and Immunity Part 1

Most encounters with microorganisms do not result in disease. The few microbes that manage to cross the barriers of skin, mucus, cilia, and pH are usually eliminated by innate immune mechanisms, which commence immediately upon pathogen entry. If phagocytosis cannot rapidly eliminate pathogen, inflammation is induced with the synthesis of cytokines and acute phase proteins. This early induced response is not antigen-specific and does not generate immune memory. Only if the inflammatory process is unsuccessful at eliminating pathogen will the adaptive immune system be activated, a process which requires several days to produce armed effector cells.

Pathogens infect specific locations in the host and damage the host by several mechanisms. Extracellular pathogens and their exotoxins are susceptible to phagocyte destruction and to antibody neutralization and opsonization. Intracellular pathogens must be eliminated by NK or cytotoxic T cell lysis of the infected cell or macrophage activation by Th1 cells. The immune response can also damage the body by means of inflammation and cytotoxicity. Review Infectious Disease for more information about common human and animal pathogens.

Except in cases of wounds, injections, or insect bites, the initial barrier to infection is the skin and internal mucosal epithelial surfaces. Besides the physical barrier of the tightly opposed epithelial cells, mechanical barriers of beating cilia, air movement, mucus, and peristalsis remove the pathogen unless it can adhere to the epithelium. Chemical barriers to microbial colonization include low pH, hydrolytic enzymes, and antibacterial defensins. Normal flora also cover surfaces and compete with pathogens for physical space and nutrients.

Once a pathogen penetrates the skin or mucosal epithelium, it usually establishes a local infection. Tissue damage and pathogen antigens (Antigens) signal tissue macrophages to secrete chemotactic cytokines (Cytokines) called chemokines to attract additional phagocytes and allow more fluid and cells to enter the tissues at the infection site. Neutrophils and macrophages engulf pathogens and destroy them. The alternative complement cascade (Complement) is activated on pathogen surfaces to promote phagocytosis and pathogen lysis. Anaphylatoxins C3a and C5a attract more leukocytes and increase capillary leakiness at the infection site, allowing phagocytes and complement to enter the tissues. Inflammation is the influx of fluid and cells that results in redness, swelling, heat, and pain (rubor, tumor, calor, dolor) at the infection site. The liver responds to macrophage cytokines by secreting acute phase proteins that further promote complement activation and phagocytosis. Macrophage cytokines also raise body temperature, signal the marrow to release more phagocytes, and activate NK cells to kill virus-infected cells. In a virus infection, infected cells secrete interferons alpha (IFNa) and beta (IFNb) to make nearby cells resistant to virus replication.

If the innate immune response does not rapidly eliminate pathogen, adaptive immune responses are stimulated. To initiate adaptive immunity, soluble antigen and APC (antigen-presenting cells: macrophages and dendritic cells) containing antigen are carried in the lymph to nearby lymphoid organs. There, antigen binds B cells and APC present antigen peptides on their membrane MHC to activate antigen-specific T cells. T helper (Th2) cells activate antigen-binding B cells to produce antibody and become effector plasma cells. Cytotoxic T cells (Tc) respond to presented antigen on virus-infected dendritic cells (DC) by becoming active cytotoxic T lymphocytes (CTL). Secreted antibody is carried by the circulation to the site of infection, where it neutralizes and opsonizes antigen and activates the classical complement cascade. Activated T cells also migrate to the infection site, where Th1 cells activate macrophages to more efficiently kill phagocytosed pathogen and CTL kill virus-infected cells. Adaptive immunity cannot be detected until 7-10 days following an initial exposure to antigen.

Migration and Adhesion

Cell adhesion molecules (CAMs) are involved in cell-cell attachment throughout the body. Immune CAMs direct leukocyte recirculation and promote leukocyte activation.

Adapted from Janeway et al. Immunobiology, 4th edition, Garland Publishing Company, New York, 1999. Fig 8.4.

CAMs allow mature T and B cells leaving the thymus and marrow to find the T and B cell areas in secondary lymphoid organs. Both T and B lymphocytes travel repeatedly between secondary lymphoid organs, blood circulation, tissues, and lymph waiting for antigen stimulation or in response to cytokine signals. On blood vessel endothelium, CAMs called vascular addressins indicate to recirculating leukocytes their location and proximity to a secondary lymphoid organ or infection site. CAMS on lymphocytes and macrophages also stabilize cell-cell binding so that antigen can be presented and cytokine and co-stimulatory signals can be exchanged. Some CAMs are expressed constitutively, while inflammatory cytokines upregulate expression of others. Examples of major groups of immune CAMs are listed in the table above.

Selectins are CAMs which resemble lectins in their ability to bind carbohydrate. Three kinds of selectins are responsible for leukocyte binding to vascular endothelium prior to their movement into the tissues: L-, E-, and P-selectins. Selectins bind carbohydrates on the mucin-like family of adhesion molecules. For example, T cells entering peripheral lymph nodes use the homing receptor L-selectin to recognize mucin-like vascular addressins on specialized cells lining the capillaries (high endothelial venules or HEV). Selectins and mucin-like CAMs are present on both leukocytes and vascular endothelium.

Integrins are leukocyte membrane CAMs which bind Ig superfamily CAMs on vascular endothelium. When T cells become activated, they lose L-selectin and acquire integrin VLA-4, which binds VCAM-1 on activated vascular endothelium near the site of infection. Effector T cells use a combination of CAMs to home to peripheral, mucosal, or skin endothelium. Integrin:Ig superfamily CAM interactions also stabilize interactions between leukocytes. LFA-1, CD2, and ICAM-3 on T cells bind ICAM-1, ICAM-2, LFA-3, and LFA-1 on APC to stabilize MHC-peptide-TCR binding; similar interactions occur during CTL-target binding (see T Cell-Mediated Immunity). Integrins LFA-1, MAC-1 (CR3), and p150.95 (CR4) are dimers that share a b chain but have distinct a chains. MAdCAM-1 has both Ig-like and mucin-like domains and binds both selectins and integrins.

Leukocytes leave the circulation and enter the tissues by a process called extravasation. Circulating monocytes can enter the tissues at any time to become tissue macrophages. Macrophages in the tissue are the first line of defense against pathogens. In response to pathogen binding, macrophages secrete chemokines that attract circulating neutrophils to the infection site. Complement activation produces anaphylatoxins which are also chemotactic; in addition, they cause the capillaries to dilate, slowing blood flow. Inflammatory cytokines also signal cells lining the capillaries (vascular endothelium) to increase CAM expression. Endothelial cells have granules (Weibel-Palade bodies) containing preformed P-selectin. In response to TNFa secretion by macrophages, P selectin is rapidly expressed on the endothelial cell membrane. Within two hours of TNFa stimulation, endothelial cells synthesize and expresses predominantly E-selectin. TNFa also stimulates endothelial cells to increase expression of ICAM-1 and ICAM-2.

The slowed blood flow in capillaries at the infection site allows neutrophils to approach the vascular endothelium. Neutrophil membrane sialic acid-containing carbohydrate called sialyl Lewis^x binds loosely to P- or E-selectins on the vascular endothelium. The neutrophils roll along the vessel as these loose associations are repeatedly made and broken. Chemokines from the vascular endothelium and nearby macrophages, as well as complement C5a and platelet-activating factor (PAF), bind receptors on the neutrophils, signaling them to activate membrane integrins by changing their conformation. Activated integrins Mac-1 and LFA-1 then bind more tightly to ICAM-1 and ICAM-2 on the vascular endothelial cells until the neutrophils stop rolling and crawl between endothelial cells into the tissues (diapedesis) using LFA-1, Mac-1, and the self-adhesive PECAM. Leukocytes secrete proteolytic enzymes to penetrate the basement membrane. Other leukocytes extravasate in a similar manner, although some of the CAMs involved may differ.

Pathogen Elimination

Innate immune responses include alternative complement activation by pathogen surface molecules, phagocytosis, interferon a and b secrretion by virus-infected cells, and Natural Killer activation. Complement activation promotes inflammation and pathogen opsonization and lysis. Phagocytes destroy engulfed pathogen in the phagolysosome. Natural Killer Cells lyse virus-infected cells, while interferons secreted by virus-infected cells make nearby cells resistant to virus replication.

Innate immunity begins with tissue damage and chemotaxis. Resident tissue macrophages respond first, engulfing pathogens and secreting chemokines. Phagocytes are attracted to the site of infection or injury by chemokines, complement chemotactic molecules C3a and C5a, and bacterial peptides such as fMet-Leu-Phe (fMLP). Macrophages and neutrophils with specific receptors for the chemotactic molecules follow a concentration gradient of chemotactic molecules towards the pathogen.

Before they can engulf pathogens, phagocytes must first bind them. Pathogen surfaces generally have sugar molecules in repeating patterns (orientation and spacing) not found on host cells that can be recognized by cells of the innate immune system. Macrophages and neutrophils have surface molecules that bind these sugars as well as common bacterial components LPS and teichoic acid. In addition, acute phase proteins synthesized by the liver link pathogen surfaces to phagocyte receptors directly or through complement. Macrophage membrane integrins Mac-1 (CR3) and p150.95 (CR4) bind several bacterial molecules including LPS, as well as complement opsonin C3b. Macrophage mannose receptor binds the common bacterial and HIV surface sugars mannose and fucose. Scavenger receptors bind sialic acid, anionic polymers and acetylated low-density lipoproteins. Some scavenger receptors also bind host erythrocyte molecules normally covered by sialic acid but exposed on old erythrocytes as a mechanism for removing old cells from the body. Phagocyte CD14 binds LPS which is coated with the acute phase protein LPS-binding protein (LBP). Encapsulated bacteria are often resistant to phagocytosis unless they are opsonized by complement or antibody.

Once antigen is bound, the phagocyte extends pseudopodia around the antigen and engulfs it, forming a phagocytic vesicle (phagosome). Cytosolic vesicles called lysosomes containing digestive enzymes at low pH fuse with the phagosomes to become phagolysosomes, whose enzymes digest the antigen. Some bacteria are resistant to hydrolytic enzymes and low pH and can escape from or even live in the phagolysosome. Pathogens are generally completely digested by neutrophils. However, macrophages also have antigen-presenting functions and preserve some pathogen peptides for presentation on their membrane Class II MHC.

In addition to their hydrolytic enzymes, phagocytes use two oxygen-dependent killing systems (oxidative burst) to kill microorganisms by oxidizing and inactivating key enzymes. Macrophages depend primarily on the peroxidase-independent system, using hydroxyl radicals (OH^-), superoxide anions (•O₂), singlet oxygen (•O) and hydrogen peroxide (H₂O₂). Neutrophil myeloperoxidase interacts with H₂O₂ plus intracellular halides to form toxic oxidants such as OCl^-. Activated macrophages also kill pathogens with nitric oxide (NO), defensin peptides, lysozyme, and secreted molecules that compete with the microbes for essential nutrients such as iron and the enzyme cofactor vitamin B₁₂. Toxic products of macrophages and neutrophils can be used inside the phagolysosome to kill the pathogen or, when the pathogen cannot be engulfed, can be excreted for extracellular killing. The latter process often results in damage to surrounding host cells as well as to the pathogen.

Pathogen binding induces other changes in macrophages besides phagocytosis and oxidative burst. A family of mammalian receptors has recently been identified that resemble a Drosophila receptor called Toll. Toll receptors in the fruit fly and similar molecules in plants trigger the release of antimicrobial peptides in response to infection, possibly representing a very ancient form of innate immunity. In mammals, at least nine Toll-like receptors have been identified. The best characterized is Toll-Like Receptor 4 (TLR-4) on macrophages. LBP-coated LPS binds macrophage membrane CD14. CD14 then associates in the macrophage membrane with TLR-4. TLR-4 signals via the transcription factor NFkB for expression of genes that encode cytokines used in defenses against Gram negative bacteria. Toll-like receptors also signal macrophages and tissue dendritic cells to express the co-stimulatory molecules B7.1 and B7.2 that are required for T cell activation. (See Receptor Signaling for details of the Toll-like receptor actions and T Cell-Mediated Immunity for details of co-receptor function in T cell activation.) Vaccine adjuvants containing microbial products have long been known to be most effective in stimulating immune responses and are now thought to work by stimulating co-stimulatory molecule expression on macrophages and dendritic cells.

If pathogens are not rapidly eliminated, an early induced response begins within four hours. The early induced response is also antigen nonspecific, but cells must be activated to synthesize effector molecules. Both the innate and early induced responses occur predominantly at the site of infection. The adaptive immune response involving antigen-specific lymphocytes and antibodies does not begin before 48 hours following antigen contact and is usually not measurable in vivo for 7-10 days. Adaptive immunity is initiated in organized lymphoid tissues near the infection site.

Macrophages are stimulated by bacterial binding to secrete inflammatory cytokines and lipid mediators of inflammation such as prostaglandins and leukotrienes. C5a, leukotriene B4, and histamine signal endothelial cells to rapidly move P-selectin from granules to the cell surface. Key inflammatory cytokines produced by macrophages include Interleukin-1 (IL-1), which activates nearby vascular endothelium to increase expression of ICAM-1 and ICAM-2 to promote leukocyte extravasation; chemokine IL-8, which activates integrins Mac-1 and LFA-1 on leukocytes to bind more strongly to vascular CAMs; Tumor Necrosis Factor alpha (TNFa), which activates vascular endothelium to express E-selectin and, with IL-8, activates neutrophils to be more cytotoxic; and IL-12, which activates NK cells to kill virus-infected cells. IL-1, IL-6, and TNFa also cause fever, a regulated increase in body temperature that stimulates immune responses and inhibits replication of some bacteria and viruses. IL-1, IL-6, and TNFa also signal the liver to produce acute phase proteins. Overproduction of TNFa in response to systemic Gram negative bacterial infection causes septic shock: systemic edema (fluid leaving the circulation and entering the tissues), low blood pressure, and disseminated coagulation that leads to multiple organ failure and death.

Two important acute phase proteins are C Reactive Protein (CRP) and Mannan-Binding Lectin (MBL). CRP binds microbial LPS phosphorylcholine to opsonize the microbe. CRP also activates C1q to initiate the classical complement cascade in the absence of antibody. MBL binds bacterial mannose and opsonizes bacteria for phagocytosis by monocytes, which lack mannose receptor. MBL also activates a serine protease cleaving C2 and C4 to initiate the MBL complement cascade. Other acute phase proteins produced by the liver include pulmonary surfactants A and D that promote phagocytosis of pulmonary pathogens by aveolar macrophages in lung aveolar fluid.

Virus infections require different effector functions, since once viruses infect cells they are protected from phagocytes and complement. Even if phagocytes could recognize infected host cells, they could not engulf anything so close to their own size. Innate defenses against viruses include the synthesis of interferons and the activation of Natural Killer (NK) cells.

Interferons a and b are made in response to virus infections by infected host cells. IFNa and IFNb are secreted and bind to membrane receptors on nearby cells; binding activates second messengers that inhibit virus replication in those cells. IFNa and IFNb also increase expression of Class I MHC to increase antigen presentation to Tc cells. NK cells are activated by IFNa, IFNb, and IL-12 to kill virus-infected cells and by IL-12 and TNFa to produce high levels of IFNg, a strong macrophage activator also produced by T cells.

NK cells are large, granular lymphocytes which are usually CD3^- and CD16⁺. The "natural" in their name comes from the fact that they are present in unimmunized individuals and they show no increased responsiveness (memory) upon secondary contact with the same pathogen. They provide an important first line of defense against virus-infected and tumor cells. NK cells have NKR-P1 lectin-like receptors which bind carbohydrates on self cells and signal NK cells to kill. NK:target cell binding stimulates the release of granular perforins that polymerize in the target cell membrane to form pores similar to those in the complement MAC and granzymes that enter the target through the pores to induce programmed cell death (apoptosis) in the infected cell. Cytotoxic T cells use the same effector molecules to kill virus-infected cells (see T Cell-Mediated Immunity).

Killing of uninfected human targets by NK cells is inhibited by Ig superfamily killer inhibitory receptors (KIR) that bind self Class I MHC. In mice, the Ly49 family inhibitory receptors are lectins which also bind Class I MHC. It is believed that virus infection alters the conformation of Class I MHC so that it can no longer deliver the "off" signal to NK cells. Virus suppression of Class I expression and promotion of cell protein glycosylation may make infected cells more susceptible to NK-mediated lysis. Understanding the details of target cell recognition by NK cells has been difficult, since NK cells have multiple KIR which each seem to recognize several Class I alleles. There is some suggestion that NK cells are selected in the thymus to recognize self Class I. In addition to killing virus-infected cells, NK cells regulate growth and differentiation of stem cells and participate in transplant rejection and autoimmunity. They also perform antibody-dependent cell-mediated cytotoxicity (ADCC, see Humoral Immunity).

Key Concepts

• Pathogens bypass physical, chemical, and mechanical barriers to enter tissues
• Macrophages recognize pathogens using receptors that bind common molecules on the pathogen surface. Tissue macrophages are in place to be the first immune system cells to see pathogen.
• Pathogen binding stimulates macrophage to phagocytose pathogen.
• Pathogen is destroyed in the phagolysosome by a variety of antimicrobial products, including hydrolytic enzymes, toxic oxygen compounds, molecules that compete for key nutrients, and defensins.
• Complement proteins are activated on the pathogen surface to promote phagocytosis, cause inflammation, and lyse the pathogen.
• Macrophages secrete cytokines that attract recirculating leukocytes to infection site.
• Increased CAM expression on vascular endothelium and on WBC allows leukocytes to extravasate from blood to infection site.
• Cytokines and C3a and C5a cause capillary leakiness and allow fluid to enter tissues.
• Macrophage cytokines also stimulate fever, neutrophil and acute phase protein synthesis, and NK cell activation.
  • Fever is a regulated increase in body temperature that slows pathogen replication and facilitates immune reactions.
  • Neutrophils are released from the bone marrow.
  • Acute phase proteins include C Reactive Protein (CRP) that binds to and opsonizes pathogens and Mannan-Binding Lectin (MBL) that activates complement.
• Intracellular pathogens are invisible to phagocytes and complement.
• Viruses stimulate infected cells to secrete Interferons a and b, which bind nearby cells and block virus replication.
• NK cells cannot bind altered Class I MHC (KIR) on infected cells, so they kill those cells by inducing apoptosis (programmed cell death).
• Pathogens that block lysosome-phagosome fusion and survive in phagocyte vesicles are protected from innate immunity.

Practice Quiz

Pick the one BEST answer for each question by clicking on the letter of the correct choice.

1. Phagocytosis must be preceded by

a. antigen binding to the phagocyte.
b. chemotaxis.
c. extravasation.
d. integrin binding to Ig superfamily CAMs.
e. oxidative burst.

2. Phagocytes bind antigen using receptors for

a. C5a.
b. chemokines.
c. glucose.
d. LPS.
e. selectins.

3. Pathogens engulfed by macrophages

a. are completely degraded by hydrolytic enzymes into their component amino acids and sugars.
b. are degraded to small peptides and carbohydrates which are presented on Class I MHC to Tc.
c. may survive and replicate in the macrophage phagocytic vesicles.
d. stimulate macrophages to adhere to B cells.
e. stimulate vascular endothelium to upregulate selectin expression..

4. An inflammatory response

a. is characterized by a decrease in vascular permeability.
b. is stimulated by cytokines produced by neutrophils.
c. occurs only during a secondary response.
d. recruits phagocytes to the infection site.
e. usually lasts for many weeks to ensure antigen is completely removed

5. Natural Killer cells

a. are stimulated to kill infected host cells via carbohydrate-binding receptors.
b. kill normal host cells with high levels of membrane MHC Class I.
c. kill virus-infected cells when the virus is acquired naturally but not by immunization.
d . recognize virus-infected cells by the presence of viral peptide on MHC Class II.
e. secrete the complement MAC to lyse virus-infected cells.

6. Interferons a and b do NOT

a. activate NK cells to kill virus-infected cells.
b. get synthesized by virus-infected cells in response to infection.
c. induce macrophages to increase expression of Class II MHC.
d. inhibit virus replication in infected cells.
e. stimulate expression of molecules required for Class I MHC presentation of viral proteins.

7. Immune system cell adhesion molecules do NOT

a. allow macrophages to leave the circulation.
b. allow T cells to home specifically to peripheral or mucosal lymphoid tissue.
c. attract leukocytes to an infection site.
d. help cytotoxic T cells to bind to their targets.
e. signal neutrophils that they have arrived at an infection site.

8. Early induced immune responses are like adaptive immunity in that they

a. are antigen-specific
b. demonstrate immune memory.
c. involve macrophages and complement.
d. involve T and B lymphocytes
e. use pre-synthesized proteins which can be released quickly upon cell activation.

9. Selectins

a. are present on both leukocytes and vascular endothelial cells.
b. bind Ig-like vascular addressins.
c. include ICAM, VCAM, and MAdCAM.
d. select antigen-specific lymphocytes to extravasate into the infection site.
e. select antigen-specific macrophages to extravasate into the infection site.

10. Lymphocyte recirculation

a. activates inflammatory cytokines to promote antigen presentation to T cells.
b. allows B cells to go to the site of infection to produce antibody.
c. circulates lymphokines efficiently throughout the body.
d. occurs for both naïve and effector lymphocytes
e. only occurs during an infection.

11. Phagocytes kill bacteria using all of the following EXCEPT

a. H₂O₂.
b. hydrolytic enzymes.
c. low pH
d. lysozyme.
e. strong reducing agents.

12. For a circulating neutrophil to reach the site of inflammation, it must bind to blood vessel endothelial cell and then pass between the endothelial cells in a process called

a. addressinazition.
b. chemotaxis.
c. extravasation.
d. marginalization.
e. opsonization.

13. Macrophages are attracted to the site of infection by all of the following EXCEPT

a. bacterial peptides.
b. chemokines.
c. C5a.
d. IL-8.
e. MAdCAM.

14. Inflammatory cytokines produced by macrophages activate all of the following EXCEPT

a. B cells to secrete acute phase proteins.
b. integrin on leukocytes to bind more strongly to vascular CAMs.
c. neutrophils to be more cytotoxic.
d. NK cells to kill virus-infected cells.
e. vascular endothelium to increase expression of CAMs.

Problems

1. Classify each of these responses as innate, early induced, or adaptive immunity.

a. A B cell activated by antigen secretes IgM specific for the antigen.
b. A macrophage processes virus peptide and presents it on MHC II to a helper T cell.
c. An NK cell binds an IgG-coated parasite and lyses it.
d. CD8+ T cells bind antigen-MHC II on dendritic cells and become actively cytotoxic T cells.
e. Chemokine blocks HIV binding to CCKR5 on macrophages.
f. Cilia and mucous carry cold viruses out of the nose.
g. Complement activated on a bacterial membrane releases inflammatory chemoattractants.
h. Complement activated by IgG on a bacterial surface releases inflammatory chemoattractants.
i. IgA blocks adhesion of Vibrio cholera to intestinal epithelial cells.
j. IgG binds pneumococcal capsule and opsonizes it.
k. IgM coats Staphylococcus pyogenes and activates complement.
l. In response to binding bacteria, macrophages increase their expression of membrane B7.
m. In response to gram negative bacteria, macrophages release IL-6 to activate lymphocytes.
n. Macrophages bind mannose on bacteria and phagocytose them.
o. Macrophages release chemokines to attract neutrophils to the site of inflammation.
p. NK cells recognize reduced MHC I on a virus-infected cells and kill them.
q. Stomach acid kills swallowed influenza virus.
r. T cells secrete IL-4 to activate B cell proliferation.
s. TNFa released by macrophages in response to binding endotoxin (LPS) stimulates the liver to release acute phase proteins.
t. Virus-activated macrophages release IL-12 to activate NK cells.

2. For each of the events in innate and early adaptive immunity listed on the left, say what the effect would be of the condition on the right.

3. Integrins LFA-1, Mac-1 (CR3) and p150.95 (CR4) share a b2 chain. Describe the immune response capabilities of a knock-out mouse for the common b2 chain.

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