Introduction

The capacity of animals to run from or disable potential predators is the essence of survival. Perhaps less apparent is the need to protect cells and tissues from attack by harmful agents—parasites, bacterial pathogens, toxins, or cells that have become cancerous.

Some everyday examples help us understand the physiological processes involved in the protection of animals from these various agents. Let us suppose that your puppy is playing in the yard, and he is splashed by your little brother as your brother rides his bicycle through a puddle of debris from the gutter. Secretions from the puppy’s eyes and mucus membranes of his nostrils act to flush material away to prevent the chance of infection. Similarly, his hair covering, eyelashes, and thick skin prevent bacteria and debris from the dirty water from gaining entrance. Even sebaceous secretions of his skin or ear canals can produce conditions that limit the potential growth of harmful bacteria. These are all examples of nonspecific defenses.

Let us suppose that your puppy simultaneously steps on a broken bottle as he runs through the puddle chasing your brother. He manages to soak the cut with dirty bacterial‐filled water. However, fortunately for him, he has received a good supply of antibodies from his mother’s colostrum. This means that he likely has specific antibodies in his bloodstream from his mother. This allows his fledgling immune system to recognize and ultimately kill bacteria present in the dirty water. This would be an example of specific but passive immunity. As the puppy grows and is exposed to various materials in his environment, he will develop his own complement of specific antibodies and the cells necessary to regenerate additional supplies of specific antibodies. Thus, defense mechanisms are divided into nonspecific and specific divisions. The immune system relies on both divisions to provide protection.

The basis for specific immunity arose from observations in the late 1800s when scientists discovered that animals that had survived a bacterial infection had protective agents in their blood (now known to be immunoglobulins or antibodies) that defended the animals against a subsequent attack by the same pathogen. It was also shown that if antibody‐containing serum from the surviving animals was given to animals that had not been exposed to the pathogen, these animals were also protected against attack. It was initially believed that the production of antibodies was the only critical requirement for protection. However, in cases where the transfer of antibody‐containing serum failed to provide protection, the transfer of the donor’s white blood cells often did provide protection. As experiments became more elaborate, it became clear that immunity involved both circulating agents and populations of white blood cells or leukocytes. Thus, the two overlapping arms of specific defense are humoral immunity (meaning blood‐derived) and cell‐mediated immunity (Rodriguez et al., 2012) (Box 15.1).

One of the major types of white blood cells is the lymphocyte. Furthermore, there are two broad classes of lymphocytes. When they are activated, B lymphocytes, usually simply called B cells, are induced to divide to generate clones of cells. You may have read or heard of polyclonal or mAbs being used in scientific experiments or more recently in some cancer treatments (Herceptin, e.g.). It is worth taking a moment to consider the difference between monoclonal and polyclonal antibodies. When the immune system in an animal is induced to produce antibodies, this is in response to multiple surveillance cells detecting the offending agent, this, then, elicits activation of multiple B cells responding to the epitopes presented for reaction. Because each of these sensing cells is likely responding to different regions or proteins or even parts of proteins or cellular components of the invader, the different activated B cells produce plasma cells that generate diverse antibodies against multiple epitopes. For example, antiserum contains multiple unique antibodies directed against the invader. Hence these are polyclonal antibodies (poly = many) generated by many different clones of B cells. By contrast, laboratory generated monoclonal (mono = one) antibodies are very highly specific and more narrowly focused on a target. This attribute can be either an advantage or disadvantage, depending on the circumstances.

Regardless, in normal whole animal immune responses, some of these clonal cells are retained as memory B cells. Other B cells differentiate into plasma cells that synthesize and secrete large quantities of antibodies. The presence of the memory B cells explains the marked increase in antibody concentration (titer) in the blood when animals are exposed to the same pathogen or antigen a second time. Essentially the pieces required for more antibody production are already in place. Cell‐mediated immunity depends on T lymphocytes, which include several subclasses of cells (helper T cells, cytotoxic T cells, and suppressor T cells). Other cells critical to functioning of both branches of the immune system include the antigen‐presenting cells (APCs) (fixed and wandering macrophages, neutrophils, and dendritic cells). For the most part, these various classes of cells have been identified based on their actions and on the fact that the cells express various classes of markers on their cell surfaces. When specific antibodies against these surface antigens are available, it allows researchers and clinicians to quantify and classify the various groups of white blood cells. Because these cells often circulate in the bloodstream or appear in secretions, it has been possible to use these markers and flow cytometry methods to identify and isolate populations of cells with specific marker characteristics. These techniques, along with cell culture and utilization of transgenic mouse models, have led to an explosion in detailed information about immune responsiveness in general and functional attributes of the different classes of leukocytes specifically. The point is that understanding of immunity, disease interactions, and physiological responses is growing exponentially. Our purpose is to provide an overview and basic ideas but like other topics covered in this text, please be aware of the rate at which current research is expanding all aspects of immunology.

While it might seem intuitive to consider innate immunity of lesser value than the specific immunity associated with antibody generation, and activation of the cells associated with cell‐mediated immunity this would be a mistake. As described by Carpenter and O’Neill (2024), many aspects of innate immunity were long ignored or neglected as immunologists focused on adaptive immunity as more important or relevant. It is now clear that both these divisions are critical. In simple terms, the innate immune system is key to recognizing and alerting the adaptive immune systems to dangers. This is analogous to having a first‐class trauma response team but no effective mechanism to transport the patient or notify the emergency medical technicians (EMTs), physicians, and nurses of the accident or danger at hand.

It is now accepted that the animal’s defense against pathogens requires multiple cells and factors in addition to the activation of lymphocytes. Moreover, these innate immunity events can elicit a kind of memory in myeloid cells that are epigenetic rather than the direct genetic alterations needed for creation of clones of B cells and generation of novel antibodies. This process has been called trained immunity to distinguish it from the generation of memory T or B cells. This recognition led to the concept that leukocytes generally have important roles in immune responses. This training memory, unlike the adaptive immune memory, which can last for the lifetime of the animal, exists for periods of months or perhaps years. This might explain how the vaccine bacillus Calmette‐Guerin (BCG), created against tuberculosis, can provide protection against a broader range of infectious agents. Support for the surveillance features of the innate immune system, exploded as researchers worked on the idea of the existence of “pattern recognition receptors” (PRRs), postulated by Janeway, (1989) as receptors that recognize “pathogen‐associated molecular patterns” (PAMPs). Since this time, a multitude of such receptors on and in various leukocytes have been discovered. These are divided into families of PRRs, including Toll‐like receptors (TLRs), NOD‐like receptors (NLRs), C‐type lectin receptors (CLRs), nucleic acid sensors, and inflammasomes. Such work confirms the concept that sensing and becoming aware of danger in an immunological sense is just as critical as having the tools (adaptive immunity) to counter these cellular and molecular dangers.

As research and interest in all aspects of immunity have expanded in the past 25 years, there have been a related explosion in identification, characterization, and naming of the cellular markers that act as immune regulators. Indeed, the title of an article by Kuśnierczyk (2024) succinctly summarizes the current situation: “Redundancy and Absurd Names in Immunology.” Some of this is to be expected as researchers independently isolate and characterize newly discovered marker molecules and, in turn, apply their own names. Here is an example. Cluster of differentiation 80 is a membrane protein expressed on the surface of many immune cells, including B cells, T cells, and especially APCs such as the ubiquitous dendritic cells. It acts as a ligand for other cluster of differentiation (CD) family proteins, for example, CD28, which together with CD80 alters T cell function. However, here is the rub. CD80 is also known as B lymphocyte activating antigen B7‐1, B7‐H1, CD28 ligand (CD28LG); CD28 ligand 1 (CD28LG1), LAB7, or BB1. There are also names that are whimsical or simply odd. For example, chemokine (C–C motif) ligand 5 is also known as CCL5, D17S1136E, SCYAS, SIS‐delta, SISd, TCP228, eoCP, C–C motif chemokine ligand 5, and finally regulated on activation, normal T cell expressed and secreted (RANTES). The molecule was first described by Dr. Tom Schall, who named the protein based on an Argentinian movie Man Facing Southeast about an alien who shows up in a mental ward. The alien’s name was Rantes, and so the acronym for the protein was made up to fit the name of the character in the movie.

This problem of different names for the same molecule is rampant in immunology research and consequently is confusing, confounding, and irritating for students and others trying to understand immunology. A similar problem exists for the naming and nomenclature of growth factors and the cascade of cell surface and intracellular molecules that explain mechanisms of action for both growth factors and hormones.

Therefore, practically speaking, the actions of the two divisions of the immune system are closely interwoven. However, generally, humoral immunity is most effective against bacteria and their toxins or free viruses because antibody binding can directly inactivate these attackers, for example, cause precipitation, agglutination, or mask surfaces, to make them susceptible to destruction by phagocytic cells or complement activation. Cell‐mediated immunity is a better weapon against cellular targets; examples include cells that have been infected by viruses, parasites, or perhaps cancer cells.

In subsequent sections, we will consider some of these elements of the immune system in more detail. However, let us begin by considering defense attributes of the immune system and physiological systems generally. Defense of the internal environment centers on three main functions (Fig. 15.1):

• Destruction or neutralization of bacterial, viral, or parasitic pathogens.
  
• Destruction of aged or damaged cells, that is, consider the finite life of red blood cells.
  
• Surveillance to detect and eliminate abnormal cells, cancerous, virally infected, etc.

Innate or Nonspecific Defenses

As the name suggests, nonspecific defenses do not distinguish one threat from another. The protection that is afforded is the same no matter what the circumstances. These measures include physical barriers, phagocytic cells, complement, general inflammatory response, and fever. The first line of defense against pathogens at first blush seems minor but the protective properties of the skin and the mucous membranes are substantial. Consider the physical barrier that intact skin provides but problems that rapidly occur if there are abrasions, cuts, or open wounds. The stratified nature of the skin epithelium, presence of desmosomes to link the cells together, keratinized surface, and secretions of sebaceous and sweat glands are highly protective. The importance of lanolin on the wool of sheep to provide protection from the elements is another example. Extend this to many animals and the added physical protection provided by fur, hair, or wool becomes apparent. The acidity of skin secretions (pH 3–5) also inhibits bacterial growth. Move to the internal passageways of the respiratory, digestive, and urinary tracts. As you should recall, the exterior portions of these tracts (oral cavity, esophagus, rectum, etc.) are also covered by stratified squamous epithelial cells. In more internal regions the epithelium is typically thinner and simple, but the cells are linked by tight junctions, which increase the barrier function of the epithelium. In addition, specialized glands (goblet cells and various multicellular glands) provide mucus and specific proteins that act to coat and protect the internal surface. Saliva and lacrimal fluids of the eye contain the protein lysozyme and enzymes that can attack bacteria. In mammary secretions produced during the nonlactating or dry period accumulation of the protein lactoferrin binds to iron, which acts to impair growth of bacterial cells. The stomach mucosa with the secretion of hydrochloric acid (HCL) and peptidases also kills many ingested microorganisms.

As a specific agricultural example, consider the importance of the epithelial layer of the teat end of the dairy cow in the protection of the internal mammary gland from mastitis (inflammation of the mammary gland caused by invading microorganisms). The teat of the ruminant has a single opening called the streak canal that leads directly into a space within the teat called the teat cistern (see Chapter 18). This means that the structure of the streak canal is critical as a primary defensive barrier against mastitis. The lowest 2 cm of the streak canal is especially important because of the capacity of tissues in this region to act as a barrier to minimize milk leakage or entrance of environmental agents. Intuitively the diameter of the streak canal is positively related to the rate of milk flow but cows with the best balance of acceptable rates of milk flow and protection from bacterial invasion have the greatest longevity in the herd. Because the teat canal is lined with longitudinal folds, dilation of the streak canal during milk causes the epithelial lining to become flattened and thin during milking. This is analogous to the changes in transitional epithelium in the bladder. Regardless, this periodic stretch allows the keratin to spread over the surface to form a bactericidal barrier. With milking some of the keratin is flushed away during the periodic opening and closing of the teat canal but fortunately, it is constantly being renewed by the epithelium (Capuco et al., 1990). The keratin itself has antibacterial agents that inhibit the growth of pathogens. Some researchers also suggest that the minute areas of secretory tissue around Furstenberg’s rosette (near the opening of the teat cistern) secrete protein(s) with bactericidal effects, but others suggest the material is lipid‐like and made by the epithelial cells producing the keratin. Reports describing the synthesis and secretion of β‐defensins by mammary cells and other immune cells support the idea that locally produced proteins likely add a layer of protection again mastitis pathogens (Daneshi et al., 2023). Certainly, the epithelial cells of the streak canal are constantly being renewed based on the appearance of mitotic cells in the basal layers of the epithelium (stratum germinativum). Passage of cannula through the teat canal or use of teat dilators scrapes away the keratin and can traumatize the epithelium. Experimentally, resistance to mastitis is markedly reduced if the keratin layer is removed. Studies in which pathogens were inoculated 3 mm into the streak canal caused infections in about one‐third of treated glands. Inoculations 4 mm into the streak canal increased infection rates further and inoculations 5 mm into the streak canal nearly always caused infection. This confirms the significance of this barrier function. Because pathogens, which cause mastitis, are not motile, to gain entrance into the parenchymal tissue they must be moved by physical forces from the outside of the teat, through the streak canal, teat and gland cistern, larger ducts, and to the alveoli. Other than the period around milking the keratin of the streak canal makes an effective barrier. However, animals with inherently thin keratin or animals with damaged teat areas are susceptible to local colonization with microbes and are therefore at greater risk for infection. During milking itself, retrograde movement of milk due to vacuum fluctuations or vacuum slips with leakage of air around the teat cups can allow bacteria‐laden droplets to pass the streak canal.

During machine milking, there are certainly dramatic effects on the teat and teat end. Given the rate of milk flow 7 to 8 m/s, it is reasonable to expect that resulting shear forces might remove some of the protective keratin. It is also probable that some milk constituents are absorbed into the keratin during the time of milking or from milk droplets remaining after milking. If milking removes substantial amounts of the keratin and if renewal is delayed or changes in composition favor the formation of bacterial colonies or adherence, this could have marked effects on the streak canal as the primary defense against mastitis.

Phagocytes are cells that engulf pathogens and cell debris. These include neutrophils and eosinophils that normally circulate in the blood and macrophages that typically reside within tissues. Some of these macrophages are considered fixed because they are permanent residents of a tissue. Examples include the Kupffer cells of the liver or the star‐shaped dendritic cells of the skin. Other “free” macrophages are more mobile and can respond to problems throughout the body or act to patrol local tissue areas. An example of the latter is the alveolar macrophages of the lung, which patrol the internal surface of the alveoli. Monocytes in the bloodstream are converted into macrophages when they exit the circulation.

Macrophages (monocytes) and microphages (neutrophils and eosinophils) share the ability to migrate and squeeze between the endothelial cells of capillaries by a process called diapedesis. This process is typically initiated by injury to the endothelial cells and/or appearance of factors that act as attractants for the cells. This is called chemotaxis. The process begins with adhesion followed by the leukocytes forming a sort of pavement of cells lined up along the periphery of the capillary. With increased permeability or damage, there is then an often‐rapid diapedesis of the cells between the endothelial cells to the region where the bacterial cells or their toxins are located. Figure 15.2 illustrates general events that occur with an inflammatory response. Many of these effects are mediated by the release of compounds produced by injured cells, proteins that appear in exudates released into the circulation, as well as molecules released by stimulated platelets and phagocytes. The local response to infection is called inflammation but depending on the circumstances, the physiological response can become more widespread. Fever, a higher‐than‐normal body temperature, occurs in response to some microorganisms and their toxins. For example, in dairy cattle, mastitis caused by Staph aureus species is typically localized but mastitis caused by E. coli is characteristically associated with marked systemic effects well beyond the appearance of abnormal milk, heat, and redness of the udder. Fever, lethargy, absence of appetite, and markedly reduced milk production are common. Neither is it uncommon for systemic problems to become so severe that physiological systems can fail, and death occurs. With marked activation of leukocytes exposed to especially potent pathogens, pyrogens secreted by the leukocytes and toxins from the microorganisms act to reset the hypothalamic neurons responsible for homeostatic control of body temperature. Prolonged high fevers are dangerous because excess heat denatures enzymes. However, milder fever is beneficial because metabolic rates of tissues are increased, allowing for more rapid repair and healing. Interestingly, while fever is known to occur in virtually all mammals, responses in birds are poorly understood (Gray et al. 2013).

When infection is severe responses can be dramatic. The creamy or yellowish pus that fills infected tissue areas is a mixture of dead cells, tissue debris, and dead or dying microorganisms. If repair mechanisms do not clear the area of debris, these materials can become walled off by layers of collagen fibers and other extracellular matrix proteins, essentially scar tissue. This is a protective mechanism, for example, isolation of the affected area, but this can also lead to the creation of an abscess. In these cases, it is often necessary to surgically drain the material before healing can take place. In some particularly difficult infections, bacterial cells can be engulfed by macrophages but not destroyed. The macrophages with their now protected residents become encased in clusters or granulomas. Since the bacteria are not actually destroyed, if the granulomas are disrupted an activate infection can resurface. This explains why tuberculosis bacilli, which are resistant to digestion by macrophages and therefore protected from antibiotic treatment, can be so difficult to treat. It is believed that some mastitis‐causing organisms behave in an analogous manner. Specifically, such organisms appear to find “protected” areas (likely walled off by connective tissue elements) so that they are safe from destruction. This could explain why some cases of mastitis are caused by the same organism repeatedly and/or some subclinical cases of mastitis.

Some of the important substances involved in inflammation and their effects are summarized in Table 15.1. Before we move to specific immunity, let us consider some of the effects of these molecules and related molecules involved in inflammation and chemical defenses. Some tissue proteins can inhibit or slow bacterial cell growth. For example, lactoferrin is a protein that is produced in secretions of the nonlactating mammary gland where it accumulates to a high concentration. This is important because it acts to bind iron that is needed for bacterial cell growth (Brock 2012). Histamine released by local mast cells at the site of an attack increases capillary permeability and vasodilation, increasing blood flow. Mast cells also produce molecules that induce chemotaxis, which act as signals to induce the migration of phagocytic cells. Moreover, the binding of these agents to receptors on the cell surface of macrophages increases Ca⁺⁺ flow into the cells. The increase in Ca⁺⁺ activates contractile elements in the cells that allow amoeba‐like movement of the cells along the gradient of chemotactic signals (Box 15.2).

As illustrated in Figure 15.3, kallikrein secreted from phagocytes cleaves inactive kininogens produced by the liver. These molecules appear in circulation and diffuse into inflamed areas because of increased vasodilation where they are converted into active kinins. Kinins subsequently stimulate several steps in complement activation, reinforce vascular bed changes initiated by histamine release, activate pain receptors, and serve as chemotactic agents to produce even more leukocyte migration and more kinin production in a positive‐feedback pattern of action. This enhances the opportunity to defeat the offending agent. These inflammatory reactions can be likened to an alarm response as a large mixture of chemicals is released into the extracellular fluids.

Myeloid precursor cells in the red bone marrow give rise to monocytes, which can progress to macrophages, or these myeloid precursors can be induced by cytokines to transition into immature dendritic cells. Macrophages generally and the specialized macrophages (Kuffler cells in the liver, Langerhans cells in the skin, and surveillance alveolar macrophages in the lungs) along with dendritic cells are especially important because these cells express proteins on their surface called TLRs. The receptors were named because they have a similar structure to a protein transcribed by the Toll gene in drosophila. They are also characterized as pattern recognition receptors and are appreciated for their capacity to recognize or bind molecules that are present on many pathogens but distinct from host surface molecules. These groupings of molecules are called pathogen‐associated molecular patterns (PAMPs).

Characterized, based on composition and structure, PRRs can be sorted into five types, based on their primary functions, cellular location, specificity of ligands involved, or evolutionary linkages (Carrol et al. 2024; Duan et al. 2022; Kawai et al. 2024).

1. TLRs
   
2. AIM2‐like receptors (ALRs)
   
3. CLRs
   
4. Retinoic acid‐inducible gene—(RIG) I‐like receptors (RLRs)
   
5. NLRs

TRLs and CLRs are membrane‐bound transmembrane proteins, and the others function as receptors within the cytoplasm. As you consider the fact that many leucocytes can be actively phagocytic and that debris from destroyed bacterial cells and infected cells can also be engulfed, the logic of intracellular receptors becomes more apparent (Sameer and Nissar, 2021).

Fundamentally the TLRs and other PRRs on these immune cells act to fill the gap between the time when innate immune cells first recognize pathogens or their components and subsequent activation of the adaptive immune response. Essentially, when these surveillance cells encounter triggering patterns, they become activated (by altered expression of surface proteins) to then engage with T cells and other cells in the adaptive immunity side of the immune system. Essentially, they become APCs. You could think of these cells as detectives reporting back to the police station to start procedures to stop a crime or provide aid to someone in need. Figure 15.4 illustrates some of the reactions involved when an activated dendritic cell engages with a CD4⁺ positive T cell.

Signaling from the family of TLRs can regulate T‐cell activation, growth, differentiation, and function in a multitude of conditions and ultimately is critical for cell‐mediated immunity. In addition, TLRs are involved in not just defense against infectious agents but in reactions to autoimmune diseases and cancer. After binding PRRs induce downstream signal cascades, for example, nuclear factor kappa B (NK‐κB), type I interferon (IFN), and inflammasome signaling pathways. These reactions result in the production of proinflammatory or antiviral cytokines and chemokines. The NK‐κB family of transcription factors is involved in immune and inflammatory responses and, for example, is important in the regulation of β‐defensin expression in the bovine mammary gland. TLR2 is activated by the lipoteichoic acid (LTA) of Gram‐positive bacteria and TLR4 is stimulated by lipopolysaccharides (LPS) of Gram‐negative bacteria.

The TLR signaling also promotes maturation of dendritic cells and induction of leucocytes needed for clearing pathogens. TLRs are part of a family of type I integral membrane glycoproteins with leucine‐rich extracellular domains and a cytoplasmic tail with a Toll/interleukin receptor domain. Combined with interleukin‐1 (IL‐1), these receptors are part of superfamily of receptors with similar attributes because all its members share the Toll‐Il‐receptor structural domain.

To the present time, 13 distinct TLRs have been identified. Each of the receptors recognizes a particular class of microbes based on the components of the bacterial cell wall. Activation of the TLR, in turn, triggers the release of cytokines. Most cytokines are low molecular weight, soluble proteins produced following activation of a “sensing” cell, for example, the macrophages with activated TLR, as described above.

Cytokine is a general term given to the chemical messengers that regulate cells involved in immune responses. However, based on genetic linkages and structural similarities many hormones and growth factors can be classified as cytokines. Interleukins are a subclass of cytokines that were first identified because they are secreted by leukocytes. At least 40 distinct interleukins are known. Classification of the various interleukins is complicated because they are grouped differently based on activity or how they interact with lymphocytes, the structural attributes of the proteins themselves, the structures of the receptors for these messengers, or if they exhibit proinflammatory or anti‐inflammatory actions. Figure 15.5 illustrates the relationship between cytokines and interleukins, actions, and properties.

Most are produced by T lymphocytes, which are identified based on their actions and expression of specific cell surface markers, especially CD4. The interleukins are key in the induction and differentiation of T cells, B cells, and other hematopoietic cells. It is telling that interleukins are also detected in other nonimmune tissues and receptors for these proteins are widely distributed. Thus, roles for these powerful signaling molecules will certainly expand in the future.

Cytokines are produced by nearly all cells involved in immunity, but the T cells are especially important. The activation of cytokine‐producing cells triggers them to synthesize and secrete their class of cytokines. The cytokines then bind to specific cytokine receptors on other cells of the immune system and influence their activity in some manner. Actions of cytokines are described as multifaceted, redundant, and pleiotropic. The idea is that a specific cytokine is likely to impact multiple target cells not just one type of cell. In addition, some cytokines are antagonistic so that one cytokine stimulates a particular action while another cytokine inhibits the same function. In other cases, cytokines act synergistically to produce a greater effect than either would have alone. These features mean that there are enormous opportunities for control and regulation of immune responses. Most of the cytokines act on leukocytes or endothelial cells to affect inflammatory responses. However, there is also an overlap between classic hormones and cytokine signaling pathways. For example, leukocytes and macrophages express prolactin receptors. It seems likely that, in addition to its well‐characterized actions in mammary development and reproduction, prolactin also acts as a cytokine in the immune system. Leptin, a hormone produced by adipocytes, is also now recognized for its impacts on the immune system (Procaccini et al. 2012). Some features of a few selected cytokines are described below.

TNFα is the major cytokine involved in acute inflammation. It is primarily synthesized by monocytes, macrophages, and helper T cells. When produced in very large amounts it is believed to be the cause of systemic shock in severe reactions. It acts on the endothelial cells to stimulate inflammation and blood clotting. It also promotes endothelial cells to secrete selectins (adhesion molecules) that are important for diapedesis of leukocytes. Furthermore, it triggers macrophages and endothelial cells to secrete chemokines. Chemokines also impact diapedesis and chemotaxis, and additionally promote macrophages to secrete interleukins (see below). Finally, TNFα is directly cytotoxic for some tumor cells, thus explaining its name.

The interleukins include a very large family of structurally similar cytokines. IL‐1 is especially significant. It has actions functionally such as TNFα (an example of some of the redundancy among cytokines). Common effects include induction of fever and sleep and stimulation of collagen synthesis as well as collagenase needed for tissue repair and remodeling. It also stimulates T and B lymphocytes to proliferate. IL‐1 is also produced by monocytes, macrophages, dendritic cells, and a variety of other cells in the body.

Other interleukins include IL‐2, which is secreted primarily by helper T cells to stimulate the proliferation of helper T cells and activate the NK T cells. It is also called T‐cell growth factor. IL‐3 promotes hemopoiesis to generate precursors of lymphocytes and mast cells. IL‐4, also produced by the helper T cells, stimulates B cells and enhances antibody secretion by active plasma cells, especially the secretion of IgE. IL‐5 behaves in a similar fashion but is more likely to promote plasma cells to secrete IgA‐type antibodies. It also acts as a chemoattractant for eosinophils. IL‐6 has wide‐ranging effects, including the promotion of differentiation of B cells into plasma cells and stimulation of the liver to secrete a mannose‐binding protein, which triggers complement protein binding to the surface of microorganisms that have mannose‐containing polysaccharides in their cell walls. IL‐8 promotes angiogenesis, an action that is clearly important in the repair of tissue damage. IL‐10 acts to dampen or turn down immune responses so it is important in the overall regulation of immune function (Mertowska et al., 2024).

Transforming growth factor β (TGF‐β) is also a suppressor of immune responses by its capacity to inhibit the proliferation and function of T cells and the proliferation of B cells. Along with the other cytokines released, TGF‐β is an important participant because of its role in several stages of wound healing. Vascular endothelial cells are early responders. There is enhanced secretion of adhesion molecules (VCAM‐1, ELAM‐1, and ICAM‐1) around the endothelial cells that gives a foundation for the anchoring of circulating leukocytes, which express receptors (integrins, selectins, etc.) to recognize these adhesion factors and allow accumulation of the leukocytes, chemotactic attraction, and diapedesis. Indeed, TGF‐β is a potent chemoattractant. Because of its effects on the secretion of extracellular matrix proteins by stromal cells (fibroblasts), it also promotes tissue repair. TGF‐β is produced by T‐lymphocytes, macrophages, and other stromal tissue cells and appears in circulation in a latent form that is activated by tissue proteases.

The colony‐stimulating factors (CSFs) are an additional group of proteins that impact immune function by their ability to induce the production of colonies of the different leukocyte types in the bone marrow. Some specific CSF members include granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF), and macrophage colony‐stimulating factor (M‐CSF). Aside from effects on proliferation the CSFs also influence leukocyte function. For example, when GM‐CSF binds to receptors on neutrophils, eosinophils, or monocytes, it activates the cells and enhances cell survival. GM‐CSF increases the capacity of these phagocytes to form pavements involved in diapedesis between endothelial cells and improves the ability of the cells to destroy engulfed bacterial cells. CSFs are produced mostly by T cells and macrophages.

A final group of cytokines to introduce are the interferons. As the name suggests these molecules were characterized by having an ability to interfere with something. In this case, it is the replication of viruses. Like other cytokines, there are multiple types of interferons (α, β, and γ). In short, as illustrated in Figure 15.6, interferon provides some resistance to viral infections by interfering with the replication of the virus in neighboring potential host cells. In addition, interferon(s) enhance the phagocytic activity of macrophages and stimulate the secretion of antibodies by plasma cells. Furthermore, these molecules markedly improve the function of NK and cytotoxic T cells, which are important in the destruction of virus‐infected and cancerous cells.

Figure 15.7 provides an overview of an essential aspect of inflammation, the migration of phagocytes to the affected area. In this example, the rapid response of PMN into the mammary gland following an experimental insult, that is, intramammary infusion of endotoxin, is shown. Since the site of the inflammation is deep within the areas of the mammary gland that store milk, the phagocytes must move out of the capillaries (between the endothelial cells) but additionally pass the basement membrane and between the epithelial cells that compose the outer structure of the mammary alveoli. In these cases, the phagocytes have responded to chemoattractant agents in milk that have diffused into surrounding interstitial fluids and local capillary beds.

As an example, the somatic cell count of raw milk is the most common dairy producer‐related method to evaluate milk quality and udder health status of individual lactating cows. Leukocytes and a small percentage of epithelial cells normally occur in milk. This combination of cells is referred to as the milk somatic cell count (MSCC). The term somatic, which means body, alludes to the fact that these are normal body‐derived cells. Most ~98% of the cells are leukocytes, and most of these are neutrophils, sometimes called polymorphonuclear leukocytes (PMN). This descriptive term is a reference to the lobed nucleus of these cells. Milk from uninfected cows typically contains less than 200 000 cells per ml and it is not uncommon to find uninfected cows with MSCC of 50 000 cells or less. Milk samples with values greater than 400 000 cells per ml are very likely from cows with inflammation most likely caused by mastitis‐producing organisms. These leukocytes enter the milk because of homing to the mammary gland from the bloodstream in response to chemicals released directly by bacterial cells or materials released by injured mammary cells. These chemicals induce chemotaxis that initially recruits neutrophils and thereafter macrophages (monocytes) into the udder. Since an increase in MSCC is closely correlated with intramammary infection, the MSCC is measured for milk samples collected as a part of routine monitoring of milk composition in many dairy herds. However, it is important to remember that strictly speaking, bacteria‐induced mastitis can only be confirmed by the isolation of pathogenic organisms in aseptically collected milk samples by approved bacteriological methods. Figure 15.8 shows the marked ability of PMN to respond to an intramammary signal. In this case, the mammary gland of the cow was infused with purified endotoxin diluted in sterile physiological saline. By the time of the next milking the MSCC has increased ~30‐fold and there is a corresponding sharp decrease in milk production. However, after several days, the cell response and milk production return to normal. This is an experimental situation, but it certainly demonstrates the dramatic response that phagocytic cells can make in response to stimulation.

Fortunately, most tissue regions have macrophages that are residents. For example, with a skin break, these resident macrophages can begin phagocytizing microorganisms (assuming they are recognized as invaders) almost immediately. However, the number of cells is limited so a full‐blow response depends on chemotaxis to recruit additional phagocytes (more macrophages and neutrophils). It is important, of course, that these cells express the ability to recognize foreign material so that normal cells are not harmed. The tagging of materials to be engulfed is complex but when it comes to bacterial cells, the complement system is especially important. This is because the effects of activated complement can be very potent but secondly presence of specific antigens and antibodies on the surface of the bacterial cells (or other materials) acts to mark these invaders for destruction by phagocytes as well as attack by complement proteins.

The complement system, usually simply called complement, is a complex of at least 20 proteins present in the circulation (originally produced by the liver). These proteins provide an important mechanism for the destruction of foreign substances because, when activated, they greatly enhance the inflammatory response but even more impressive they can stimulate the direct destruction of bacteria and some other cells by causing the cells to rupture. Aside from direct effects on bacterial cells, activated complement also enhances inflammation in several ways.

• Stimulates release of histamine from mast cells.
  
• Promotes vasodilation and thereby increases vascular permeability.
  
• Activates kinins.
  
• Coats the surface of microorganisms and acts as an opsonizing agent.

Thus, while complement is nonspecific, it clearly enhances or “complements” the immune response, hence the name. The effects of complement on bacterial cells are illustrated in Figure 15.9.

Specific Immunity

Despite the impressive benefits of non‐specific defenses, this alone is not sufficient. Hallmarks of both cellular and humoral immunity include (1) specificity, (2) systemic rather than local responses, and (3) evidence of memory. Specificity indicates that the immune response is directed at a unique antigen. Systemic responsiveness refers to the notion that a response to an attack can be mounted regardless of the point of entry. Memory indicates that the immune system is better prepared with a faster response when exposure to an antigen occurs a second time. This is the idea behind vaccinations. A vaccination is essentially the induction of a specific immune response and creation of immunological memory because of a planned exposure to the antigen in a manner that does not cause illness. For example, immunization with cell wall components or killed bacterial cells (incapable of causing disease) can nonetheless induce an immune response because of the foreign proteins or polysaccharides present in these preparations. If the animal is then exposed to live microorganisms, the animal can respond more quickly so that the chance of exhibiting the disease is reduced or symptoms are milder and less severe compared with nonimmunized animals.

Let us first consider humoral or antibody‐mediated immunity. However, it is worth remembering that the two divisions of the immune system (humoral and cell‐mediated) do not function independently but rather together to enhance protection. As shown in Figure 15.10, it is also important to realize that immunity can be either passive or active. Clearly, the protection provided to the newborn calf (or puppy in our earlier example) by suckling colostrum from its mother is critical, but it is limited. The antibodies provided in the colostrum are only a stopgap measure until the calf or puppy can begin mounting its own immune responses with related immunological memory.

Antibody Structure and Function

There are five major classes of immunoglobulins: IgA, IgG, IgM, IgD, and IgE. IgG is the most abundant and diverse antibody in circulation. It accounts for ~80% of the total. It is primarily responsible for both primary and secondary antibody responses (increased blood titers) following immunization or other exposure to antigens. IgG also circulates as a monomer. IgA also appears as a monomer but is limited in the circulation. It occurs more frequently in secretions (saliva, sweat, intestine, etc.) associated with mucous membranes and epithelial surfaces. It also is most frequently secreted as a dimer with the two antibody molecules joined by a third element called the secretory piece. IgM appears as a monomer and in groupings of five antibodies (pentamer) linked together. In the monomeric form, the antibody is usually attached to the surface of B cells. During primary antibody responses, the pentamer form of IgM is the first class of antibody released by the plasma cells. Because there is usually only a small amount of IgM free in circulation, detection of an increase in the blood is diagnostically useful as an indicator of a current infection in an animal. IgD is nearly always attached to the surface of B cells where it functions as a receptor for the activation of the cells. IgE is secreted by plasma cells in the skin and mucosal membranes. When the IgE is bound to antigen, the stem region of the molecule reacts with mast cells and basophils, causing them to release histamine and other chemicals. Unfortunately, hyper or inappropriate IgE‐induced activation of these cells is involved in many allergic reactions. This explains the significance of antihistamine treatments in treating allergic reactions.

Whatever it’s specific class, each antibody molecule has a basic structure that consists of four protein chains linked together by disulfide bonds. Two identical heavy chains of about 400 amino acids each make up the bulk of a structure that resembles the shape of the letter Y. Two additional identical protein chains (the light chains) essentially overlap the portions of the heavy chains in the arms of the Y and are joined by sulfide bonds. The heavy chains have a hinge‐like region near the top of the stem of the Y. The two ends at the top of the letter Y of the antibody molecule create the sites for binding of the antibody to its antigen. This means that the antibody is divalent; that is, one antibody molecule is capable of binding two antigen molecules. The stem region of the molecule is significant because it contains sites for complement binding and macrophage activation. These sites are important because the binding or fixing of the antibody allows the development of a cascade of reactions important in the immune attack. The structure of an antibody molecule is illustrated in Figure 15.11.

Although antibodies do not directly destroy pathogens, their binding to antigens on the surface of bacterial cells, for example, marks the cells for destruction. Antibody binding to toxins or foreign debris can also inactivate these agents by neutralization, precipitation, or, in the case of cell‐associated antigens, agglutination. These reactions greatly enhance inflammation, along with inflammation‐induced chemotaxis and recruitment of leukocytes that destroy bacterial cells by phagocytosis. Antibody binding also triggers complement fixation and exposes the macrophage‐binding sites on the antibody molecule. The coating of foreign substances with antibodies is called opsonization.

Antigens are substances that can activate the immune system thereby provoking an immune response. Most often these are large, complex molecules (typically proteins) that do not normally appear in the body. In other words, the immune system does not recognize them as self. In the case of completely reactive or immunogenic antigens, these molecules induce the proliferation of specific lymphocytes and the synthesis and secretion of specific antibodies. In other cases, many small peptides and nucleotides are not immunogenic in themselves, but when linked with other self‐proteins the new combinations can produce dramatic even harmful responses. This is the basis of some allergenic responses. Researchers were able to take advantage of this property to create antibodies against normally nonresponsive molecules, that is, steroid hormones to create many immunological‐based assays, for example, radioimmunoassay, enzyme‐linked immunoassays (ELISA), and western blotting.

B Cell Selection and Antibody Secretion

When B cells are stimulated by antigens a cascade is initiated so that antigen binding to surface receptors on a (naïve or previously inactivated B cell) becomes activated to complete its differentiation cycle. This activation process, usually in combination with T cells, triggers clonal selection and expansion. The initial step induces B cell growth followed by rapid proliferation of an army or clone of identical daughter cells all of which express receptors specific to the antigen that initiated the process. Since all the cells are identical, they form a group called a clone. In cases where a particular antigen leads to the production of a single clone of cells, this would be called a monoclonal response. This situation is taken advantage of experimentally to produce highly specific mAbs. More often in physiological situations, several different families or clones of stimulated B cells are generated. Because each of the antibodies that are produced is likely to recognize different epitopes of the same antigen, these antibodies are referred to as polyclonal antibodies.

Most of the stimulated B cells are induced to become plasma cells. This is fortuitous because plasma cells have an extensive array of rough endoplasmic reticulum (RER). Thus, the capacity of these plasma cells to synthesize and secrete antibody molecules (proteins) is very high. Antigen stimulation of B cells is illustrated in Figure 15.12.

In the primary stimulation, antigen (green spheres) binds to receptors on the surface of selected B cells. This induces cell proliferation and production of clones of identical cells. Some of the cells enlarge into B lymphoblasts and then plasma cells that secrete antibodies specific to the antigen. Other clonal B cells remain as memory B cells. With a subsequent exposure to the antigen (weeks, months, or even years later), a second more rapid induction and secretion of antibodies and generation of additional memory B cells occurs (Rawlings et al., 2012).

It is estimated that each plasma cell can secrete more than 2000 antibody molecules per second. These antibodies have the same antigen binding capacity as the receptor proteins on the surface of the B cells that first bound the antigen. Clonal B cells that are not activated to become plasma cells become long‐lived memory cells. It is the presence of the memory B cells that explains the very rapid and sustained secretion of antibodies that occurs when an animal is exposed to an antigen for a second time. This pattern of response is illustrated in Figure 15.13.

The blue line illustrates the relative antibody secretion response to immunization with antigen X. The initial response begins after several days, peaks at a relatively low level, and declines markedly by four weeks. A second exposure to antigen X elicits a rapid and relatively much greater response. Exposure to a second antigen Y at the same time has no effect on the response to antigen X and the relative response to antigen Y is like the initial reaction noted to immunization with antigen X.

Cell‐Mediated Immunity

Antibodies are extremely important, but it is also clear that effectiveness depends on the capacity of the antibodies to recognize specific pathogens. How are our animals protected against viruses or infections from microorganisms that can “hide” from detection by antibodies? The T cells provide this added more complex layer of protection. Two primary groups of T cells have been recognized based on the complexes of glycoproteins that are expressed on their cell surfaces. These are CD4⁺ cells, also known as helper T cells (T_H), and a larger population the CD8⁺ cells that include cytotoxic T cells (T_C). Some of the CD8⁺ expressing T cells are also suppressor (T_S) cells that act as modulators of cell‐mediated immunity.

While B cells and antibodies bind to and respond directly to antigens, T cells do not have this ability. Instead, the T cells can recognize and respond to pieces or fragments of protein antigens that have been processed by other cells of the immune system, the APCs (macrophages, neutrophils, dendritic cells of the skin, etc.). This process is illustrated in Figure 15.14.

Cytotoxic T cells (T_C), also called killer T cells, can directly attack and kill other cells. When activated these cells migrate through the circulation and the lymphoid tissues seeking cells that express antigens for which the T_C cells have been sensitized. The primary targets of the cells are other cells that have been infected by viruses but under some circumstances, they can attack cells that are infected by bacteria or parasites. They can also act on cancer cells and are the primary cells involved in transplant rejection reactions (Qu et al., 2024).

Before the cells can respond, the T_C cells must link or dock with potential targets by binding them to the self/nonself complexes on the cell surface. Briefly, the surface of all cells expresses a myriad of proteins. However, if the immune system has been appropriately programmed all these self‐antigens are not recognized as foreign by the animal but are strongly antigenic to other animals. This is the essence of blood transfusion or graft rejection between unrelated animals. Some of the major surface proteins are part of a group of glycoproteins called major histocompatibility complex or MCH proteins that are coded by major histocompatibility complex (MHC) genes. Because there are virtually millions of possible combinations for the complex of genes that code for these proteins, except for identical twins, it is very unlikely that two animals would have expression patterns that would be the same between individual animals. It is even more complex in that there are two major clusters of MHC proteins and genes. Class I MHC proteins are expressed on surfaces of essentially all cells, but class II MHC proteins appear only on certain cells of the immune system. Each MHC protein has a cleft or groove that displays a peptide. In normal cells, the peptides that are displayed in this cleft are peptides that are derived from the normal recycling of cellular proteins. When some cells become infected or are altered by cancer the MHC proteins can bind and display peptides derived from bacterial cells, viruses, or cancer‐mediated processes. This then acts to mark the infected or cancerous cells as nonself thereby targeting it for close surveillance and possible attack by the T_C cells.

Essentially the CD8⁺ class of lymphocytes or T_C cells is activated when they dock with other cells that have processed antigens in combination with the class I MHC proteins that are not recognized as self. The example shown in Figure 15.15 illustrates the activation of T_C cells that have encountered a virus‐infected cell and consequently a clone of activated T_C cells is produced. These cells can then detect and bind to other infected cells. The fundamental result is that this encounter induces the T_C cell to divide and produce clones of identical cells. Some of these new cytotoxic T cells become memory T cells while others are activated to seek out cells that exhibit the antigen/MHC on the cell surface that initiated the process in the beginning. Another class of cells the NK cells is discussed in Box 15.3.