The Immunology of Large Animals



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The Immunology of Large Animals
David J. Hurley, PhD

Associate Professor of Population Health and Large Animal Medicine, and

Molecular Microbiologist, Food Animal Health and Management Program

College of Veterinary Medicine

University of Georgia

Athens, GA 30602



Abstract


Immunology is generally taught as if all animals shared a uniform set of mechanisms to effect the protection of the body and regulate those processes. Most of current immunology has been “established” by studies conducted with mice. Mice have a unique set of environmental and evolutionary challenges not shared by all other animals. We know that cattle, swine and horses are not just “big mice” in other ways, and it turns out that they are not immunologically identical with mice (or each other) either. This essay will summarize the differences between the immunology generally taught to veterinary students and how cattle, swine and horses mount and regulate inflammatory and adaptive immune responses.

Introduction

Thus far in your training, you have learned immunology based primarily on the lessons taught to us by mice and as applied to human conditions. To really appreciate the complexity of the infectious diseases of large animals, their diagnosis and their treatment; it is important for you to see the outstanding features that guide the response these animals make to dangerous and damaging insults, such as the tissue damage caused by colonization and infection, metabolic and structural products of invaders, and how the ecology of each host has directed the process of response on the innate, intermediate and adaptive levels.

Understanding the relationship between immunology and the diagnosis and treatment of disease is a critical factor in being an informed physician. As a veterinary physician, you must have a feel for the factors that drive and control the responses of each group of animals to the dangerous and damaging events in their internal environment. Large animals are a particularly interesting group. They represent, in my opinion, the greatest diversity in the control of innate immunity and range in dependence on adaptive immunity to resolve problems that one has the opportunity to study in all of medicine. The contrast between cattle and horses in the control of inflammatory function and use of intermediate response (between direct pathogen associated molecular pattern (PAMP) recognition and major histocompatibility complex (MHC) restricted) mechanisms is the most clearly defined in all of medicine. Understanding these differences will instruct you in ways to approach the treatment and care of these animals.

Most of the immunology we teach in introductory classes, either in undergraduate school or to veterinary and medical students, is based on studies conducted with mice. This is for good reason. We have the largest body of data from studies with mice. They are small and inexpensive to use, we have generated a tremendous number of genetically defined and knockout variants for use in reductive study of specific pathways, and there are many research tools available for purchase or exchange to assist us in doing our studies. In presenting the complex field of immunology to students for the first time, we attempt to teach a family of “general rules and principals”. This helps the student to organize and understand the processes and responses more easily, and to connect the protective and damaging aspects of the same families of responses more clearly in their general framework. (For example, understanding the relationship between antibody-complement immune complexes in bacterial clearance and the development of type III hypersensitivity responses as two sides of one coin). One caveat for the student is that all animals (including humans) are not just big mice.

Why should we learn about the immunology of large animals? Large animals represent several families that have evolved under different sets of ecological pressures. Most obvious are the ruminants. Cattle and their relatives carry a large, shedding bioreactor within their body cavity that presents huge quantities of PAMPs on a regular basis to indicate regular danger. Cattle have adapted the most exquisite inflammatory control of any animal it has been my honor to study. Horses faced different pressures. Much better contained fermentation systems for extracting nutrition from cellulose than cattle, but highly responsive inflammatory activity on sensing danger. The most difficult and feared diseases of the horse, colic, chronic obstructive pulmonary disease (COPD) and laminitis, all appear to have significant systemic inflammatory components in their pathogenesis. Swine were domesticated to live on garbage. They are low to the ground and eat a highly varied diet and raise a lot of dust finding it. These are all factors that should instruct our study of their diseases.
Of mice and not men – diversity in immunity

There was a great review of the comparative immune activity of mice and men published by Metas and Hughes in the Journal of Immunology (J. Immunol. vol 172, pp 2731-2738, 2004). The authors compiled a list of differential features of the mouse and human immune system for those of us who are really into immunology. If you chose to read it, and I do recommend it to you, you will find it assumes you are highly conversant with modern immunology. The vocabulary and intensity of the paper are at a quite high level. Yet, the major points will be clear to you as you review it. (The basic areas they address were covered in your immunology courses). What is their point? Well, they are trying to show us that we must be careful in making assumptions about immunological assessments and treatments developed in mice, as they may not translate directly into the same outcomes in humans (or in our case large animals). They point out significant differences in every aspect of immune system function and regulation between mice and humans including: hemopoesis, the use of toll-like receptors (TLR) in PAMP recognition, recognition of antigen, the intermediate immune response, chemokine usage in staging immune function, and how grafts are tolerated and rejected. The table in their paper is packed with great information. Probably too dense for your everyday purposes, but what you may eventually need to find someday is likely at least hinted at there.

Mice and humans have big differences in their approach to inflammation. Mice have only about 10% neutrophils in circulation, but humans have about 45%. Mice tend to deal with dangerous invaders using tissue macrophages and recruiting monocytes to the tissues. Humans recruit neutrophils, which in turn recruit monocytes to the site of danger or damage. The use of specific members of the TLR family of receptors is also different in mice and humans, with the greatest differences observed in the distribution of TLR 2, 3 and 9. Defensins are also ecologically different between mice and humans. Mice store pro-enzymes, humans store active proteins, and mice do not produce defensins in leukocytes.

Mice and humans also have big differences in control of Th1/Th2 polarization. Mice rapidly and permanently differentiate lymphocyte responses into Th1 and Th2 contexts. This polarization is maintained in memory cells and provides a fixed reactivation context. This is not observed in humans. While, Th1 and Th2 response contexts are measured in active responses, the context is more plastic and memory reactivation can have a Th1, Th2 or Th0 bias. The role of IL-10, an anti-inflammatory cytokine, is also different between mice and humans in that IL-10 is produced in humans as part of both Th1 and Th2 response, but only in Th2 responses by rodents.



Differences in antigen presentation are also observed between mice and humans. Antigen presentation by epithelial cells is observed in humans, but only presentation by dendritic cells, macrophages and B cells have been observed in mice. It also appears that activated T cells can participate in antigen presentation through MHC class II in humans, but never in mice. These enhanced antigen presentation capacities in humans also appear to be related to differential presentation of CD40 on epithelial cells and T cells not observed in rodents.

Table 1. Comparison of the Immunity of Mice and Men

Feature

Mouse

Human

Percent neutrophils in blood

10

45

Percent lymphocytes in blood

75

40

Percent monocytes in blood

10

10

Percent gamma-delta in blood

2

2

CD4:CD8 ratio in adult

2:1

3:1

Polarization of Th1/Th2

very strong

moderate

NO production to LPS

very strong

moderate-weak

ROS production to LPS

weak

moderate

MHC class II expressed on T cells

never

activated

epithelial cells present AG

no

yes

Predominant panT cells CD

3

2,3

Transplasental immune transfer

major

moderate

Immunoglobulin classes

IgG1,2a,2b,3

IgG1,2,3,4




M, E, A

A1,A2, M, E, D

Light Chain usage

kappa

Both used

Secretory Ig

A

A

lymph

cellular

cellular

lymph nodes

normal

normal

Humans and rodents each produce a unique set of chemokines. Chemokines appear to act as the “vote” each local environment offers to inform the secondary lymphoid tissue about the state of danger and damage it is experiencing to inform the overall response to danger in the regional and systemic response. Humans produce CXC receptor 1 and the ligands CXCL8 (IL-8), CXCL7, CXCL11, CCL13 (MCP-4), CCL15, CCL18, CCL23, and CCL24/26 (eotaxins 2/3). These have all been identified in humans and play critical roles in disease states, but have not been identified in mice. In contrast, CCL6, CCL9, CXCL15 (lungkine), and CCL12 (MCP-5) play a role in mouse infectious diseases, but have not been identified in humans.


Immunology of cattle (including goats, sheep and water buffalo)

The rumen is an ecological factor in cattle. The rumen produces huge quantities of microbes in the digestion of grasses, and products that represent PAMPs escape the rumen environment and insult cattle on a regular basis. Therefore, cattle have evolved very tight regulation of innate inflammatory responses. This can be readily observed after field surgery for hardware disease. The operation is often done in the mud and the surgical wound closed, then the animals released to the field. The next day or the day after, the wound is in the process of healing and the cow eating happily. This is a truly remarkable sight.

Cattle have a balance between neutrophils and mononuclear cells in circulation. The inflammatory cells produce both nitric oxide (NO) and radical oxygen species (ROS) in moderate concentrations after stimulation in vitro with LPS (particularly from Salmonella). It appears that both NO and oxygen radicals play important roles in direct inflammatory control of dangerous invaders in cattle. Cattle also appear to rely on intermediate immune responses (regulated by lipid and phospho- or lipo- protein) for control of infection to a much greater extent than mice or humans. Cattle have a relatively high number of circulating gamma-delta cells. These cells appear to recognize antigens in the context of CD1 antigens, similar to men. Three different CD1 antigens have been identified in cattle, CD1d, a probable CD1b and a possible CD1c that can provide the context for intermediate responses. This dependence on immune responses that are not PAMP dependent, but directly recognize structures produced by dangerous invaders may be part of the method cattle use to regulate inflammatory responses and minimize damage caused by regular exposure to microbial products.

Another example of the highly regulated inflammatory response of cattle is their tolerance of a wide variety of strong adjuvants. Many different formulations of adjuvant, including those that cause significant side-effects in other species, have been successfully used in commercial products for cattle. These include very potent oil in water and water in oil formulations containing multiple components that lead to a very broad and lasting immune response in cattle. Other evidence can also be seen in the generally well-tolerated vaccines produced as crude bacterins used in cattle. While the level of lipopolysaccharide in many of these products has been documented to cause problems in some individual animals, large numbers of cattle have been successfully vaccinated with these products to induce short-term immunity to specific bacterial diseases, like blackleg.

Cattle are more like humans than mice in the differentiation of Th1/Th2 responses to antigen. Cattle can be shown to mount specific responses in the context of Th1 or Th2, but it does not appear that memory responses are locked into these response patterns. Adaptive, classically MHC restricted, responses also appear to play a “completing”, rather than central role in the management of infections.

Immunoglobulins of mice and humans are different (see table 1). Cattle have features similar to both mice and humans, but some unique features as well. Cattle, like mice have a very high representation of a single light chain gene used in antibody (about 95%). However, unlike mice that use primarily kappa, cattle use lambda. In addition, cattle utilize IgG1 as a secreted immunoglobulin in addition to IgA. Unlike other animals, they make a secretory chain for monomeric IgG1 that is related in structure to that for IgA. Cattle also have a unique FcR that recognizes only IgG2b and is important to control of bacterial infections.



Antigen presentation presentation in cattle appears to be a complex process. The tightly regulated inflammatory responses in cattle, significant level of MHC class II expression on epithelial cells, T cells and B cells, and expression of several CD1 epitopes by bovine mononuclear cells suggests that multiple pathways of antigen presentation are occurring in concert. Development of novel and complex adjuvants for cattle vaccines were driven by the empirical recognition of this complex set of processes. Further, cells that appear to recognize virally infected autologous cells, but are not dependent on MHC class I antigen presentation for killing, have been documented in vaccinated cattle.

Table 2. Comparison of the immunological features of cattle, swine and horses

Feature

Cattle

Swine

Horse

Percent neutrophils in blood

50

40

80

Percent lymphocytes in blood

30

35

15

Percent monocytes in blood

5

5

5

Percent gamma-delta in blood

15

20

~1

CD4:CD8 in adult

3:1

0.7:1

2:1

Polarization of Th1/Th2

weak

weak-moderate

moderate

NO production to LPS

moderate

Moderate

weak

ROS production to LPS

moderate

moderate

very strong

MHC class II expressed on T cells

activated

always

Activated#

epithelial cells present AG

probably

probably

unknown

Predominant panT cells CD

2,6

2

2

Transplasental immune transfer

none

none

none

Immunoglobulin classes

IgG1, 2a,2b

IgG1, 2a,2b, 3, 4

IgGa,b,c,(B), (T), Tb*




M, E, A

M, A,E

M, A, E

Light chain use

lambda

Both

both

Secretory Ig

G1 and A

A

A

lymph

cellular

acellular

cellular

lymph nodes

normal

inverted

normal

# not universally accepted

*also called IgG1,2,3,4,5 &6 in horse








Finally, because of the structure and thickness of the placenta in cattle, no maternal immune components appear to be transferred during development to the fetus. The placenta is an absolute barrier, excluding proteins and cells. Therefore, colostrum becomes the critical link between maternal immune protection against local environmental antigens and the neonate. Failure of passive transfer is a critical problem in cattle, and an important management issue in rearing both beef and dairy calves. Maternal cells also appear to play a role in the transfer of immunity to the calf. These cells can traffic across the gut of the calf during the first 8-24 hours after birth and have been shown to travel in the neonatal circulation. Maternal cells (based on expression of maternal MHC class II antigens) have been shown to home to the gut and secondary immune tissues of the calf beginning about 12 hours after birth. Finally, it has been shown that enhanced antigen presentation, response to alloantigens (foreign MHC) and viral antigens are observed in the circulation of the neonatal calf concurrent with the circulation of maternal cells between 12 and 30 hours after receiving colostrum.


Immunology of the pig

One of the most striking features of the immune system of the pig is the low ratio of CD4:CD8 T cells, generally less than 1:1 in adult. Mature pigs also have a large population of CD4, CD8 double positive T cells in circulation. These represent “experienced” T cells and may represent the circulating activated and memory populations. The double positive population of T cells in the pig increases after infection or vaccination. In addition, MHC class II antigens are always expressed on porcine T cells, unlike most other species. The level of expression increases on activated cells in the pig and is a good indicator of an active immune response. Pigs show moderate polariztion of adaptive immunity into Th1 and Th2 responses, with considerable Th3 type responses reported.

Pigs have what has been called an “inverted” lymph node structure. That is most immune cells enter and leave the lymph nodes in through the blood. Thus, pigs have nearly acellular lymph. This makes pigs an interesting biomedical model, as they represent a unique class of animals in which the mucosal leukocytes circulate in the blood and can be monitored (against the population of central circulatory compartment leukocytes) in peripheral blood samples. For this reason, the number of circulating leukocytes in pigs is generally higher than most other animals. It also means that there is a high fraction of gamma-delta cells circulating in the blood of pigs. When they are young, as many as 35-45% of circulating leukocytes can bear gamma-delta TCR. Further, adults have about 20% gamma-delta cells circulating in the blood. So far, only a CD1a like antigen gene has been identified in swine, unlike cattle where three different CD1 genes were found. This CD1 isoform is consistent with presentation to gamma-delta cells in a fashion similar to that described for humans.

The pig also has a balanced inflammatory response that is very well regulated. Swine are good responders to environmental antigens and produce high levels of antibody and cell mediated responses to antigens. Many of the same adjuvants that were developed for use in cattle have been successfully applied to vaccine for swine. There is some evidence for the replication of gamma-delta cells in cattle and swine after infection or vaccination, and that some form of memory based on clonal expansion may be present. These finding have not yet been reported in other species.

In response to in vitro stimulation with lipopolysaccharide a balanced production of both NO and ROS is reported in pigs. Analogous to cattle, this may help to explain the tight regulation of inflammatory responses in the pig, and the capacity of the pig to tolerate vaccination with strong adjuvants. Pigs also have a moderate number of neutrophils in circulation, similar to the human. The balance between neutrophil and mononuclear cells driven inflammatory responses may also be an important factor in the balance and regulation of inflammation in the pig.

Compared to cattle and horses, pigs utilize a much more restricted selection of Vh genes to develop their repertoire of antibodies. They have a more diverse set of gamma heavy chains than cattle, but fewer than horses, with a split into IgG2a and 2b, similar to rodents, and 4 gamma chain classes like humans have been documented.

Similar to cattle, pigs have no transfer of immunity to the fetus across the placenta. It is too thick and forms a barrier. Colostrum is again the primary source of transfer of immunity to neonatal piglets. We will discuss the development of immunity in neonatal pigs during a future class period in detail.

Immunology of the horse

The immune responses and regulation in the horse make a very interesting study. I have been studying the inflammatory and immune responses of horses for about five years now, and have found that everything is more difficult to study in the horse. Horses mount strong and persistent inflammatory response to signals of danger or damage. Their circulating leukocytes are loaded with neutrophils, and up to 90% circulating leukocytes can be neutrophils in the blood, 70% is typical. In addition, unlike many other animals where monocytes and eosinophils characterize recurring inflammatory responses, neutrophil recruitment in COPD is the classical mechanism observed, and neutrophil activation in the circulation and neutrophil trafficking into the laminae are observed in black walnut induced laminitis. This is consistent with the dominant role of neutrophils in the horse. Horse leukocytes produce massive quantities of ROS when stimulated with LPS, but essentially no detectible level of NO by activation of iNOS. This does not discount the role of monocytes and macrophages in the protection of horses or in inflammatory disease. Monocyte products, such as tumor necrosis factor and IL-1 play important roles in colic and the response to endotoxin in horses. One must simply not discount neutrophil driven mechanisms when considering the pathogenesis of diseases with an inflammatory component in horses because of their dominating presence.

Another aspect of the strong inflammatory responses of horses is the problems they encounter after exposure to adjuvants. Many otherwise interesting vaccine candidates for protection of horses are eliminated before production because of side-effects encountered in testing. Adjuvants used for other large animals have often proven too strong for horses. We are working on new classes of adjuvants to better utilize the inflammatory responses of horses in development of protective immunity at UGA. These constructs are designed to spare the inflammatory consequences of vaccines. This work is just beginning, but we have some candidates in development.

The inflammatory responses of horses are strong and prolonged. A role for intermediate responses has not been well documented in horses. Lymphocytes that lack CD4 and CD8 receptors have been identified, and a role for gamma-delta lymphocytes has been postulated for horses. No CD1 antigens have been characterized in horses, so the mechanism of antigen presentation is not clear for intermediate responses.

In contrast, the classically MHC restricted T cell responses of horses have been clearly demonstrated and the polarization of Th1 and Th2 responses of horses appear to be stronger than in humans, but less fixed than in rodents. Studies of MHC restricted killing by horse lymphocytes and studies of cytokine message production support a strong role for MHC restricted responses in control of infection and a polarized memory response. A somewhat limited number of tools to assess equine lymphocyte subpopulations have impeded the complete description of equine adaptive immunity.

Horses have a unique distribution of immunoglobulin classes. A family of six gamma constant region genes, with no pseudogenes identified, has been described. It appears that horses have six classes of IgG, and renaming them IgG 1-6 has been proposed. The molecular biologists have also found evidence for at least two classes of IgA genes in horses. At present, we do not have the tools to test this fully, but it is likely that a split, similar to that found in humans, has occurred in horses. It is clear that immunoglobulin plays an important role in the defense of horses and division of that defense has occurred in the development of the large number of immunoglobulin classes that they produce.

As with the other large animals reviewed here, horses do not transfer immunity across the placenta to the fetus. Colostrum is a critical source of immunity and immune development cues to the neonatal foal. Failure of passive transfer is a critical problem for horses, and leads to significant problems in the health of the foal. Tests for failure of passive transfer should be included in a foal workup for neonatal infection. Our group has recently started to work on other factors, in addition to antibody, that are important in the transfer of maternal immunity to the foal. We have examined the roles of soluble CD14 (an LPS transfer molecule and stimulator of immune cell development) and lactorferrin on the function of leukocytes from presuckle foals. We are also studying the role of transfer of maternal leukocytes to neonates in our laboratory. It has been observed that foals are deficient in their ability to produce interferon gamma for about a month after birth. This suggests that they develop the capacity to mount T cell mediated responses to intracellular pathogens more slowly than many other species. We hope to eventually develop a better definition of failure of passive transfer for use in the clinic.

Common immunological features of large animals

Two striking features of the immunology of large animals were presented here. First, all the large animal families discussed in this essay were absolutely dependent on colostrum for the passive transfer of immunity and cues from the mother for the development of the neonatal immune system. This is radically different from mice and men where very significant transfer of immunity occurs before birth. Mice are born with much more complete immune protection and a network of responses that look much like the adult, only on a smaller scale. Humans receive about 30% of the antibody transfer from their mothers before birth and there is evidence of some cellular transfer during fetal life.

Second, all these families of large animals have some unique features in the function of antibody. Cattle have unique Fc receptors directed at IgG2b to protect the tissues and a secreted form of IgG1. Pigs have a unique system for the generation of diversity among large animals, using fewer recombinant sites in the building of an antibody arsenal. Horses have six classes of IgG and probably at least two classes of IgA. This suggests that the production and regulation of antibody is linked to the ecology of the threats to each animal and should be considered in development of vaccines and treatments for disease.
Take home messages

First, this essay represents my viewpoint. It is not a complete and exhaustive review, but a discussion of points that I believe are important and interesting. Good reviews of food animal and equine immunology can be found in many places, and I recommend Vet Clinics in N. Am. Food Animal Practice,Vol. 17.3, 2001, and Vet Clinics in N. Am., Equine Practice, Vol. 16.1, 2000 to you for further reading. The topic is far too broad and complex to be covered in a few pages. Second, I intend this essay to serve as the basis and background for a discussion of the immunology of large animals among us. I recommend you to address the questions that follow in your preparation for class. The class will be structured around my interest in comparative immunology and my “framework” for understanding the ecology, evolutionary pressure and biology of differential immune responses. The lecture will be very different from this presentation, much less formal.





  1. How does the immunology of cattle (or swine or horses) impact the types of diseases that affect cattle (or swine or horses)?

  2. How does the role of the inflammatory response differ in its impact on the pathogenesis of disease in cattle, swine and horses?

  3. How does an understanding of the immunology of each family of large animals instruct the development of methods to diagnose disease?

  4. How can information about the specific class of antibody response in horses (or cattle or swine) provide significant prognostic information to the veterinarian?

  5. How does the gamm-delta T cell response (and NK and NK-T cell response as well) act as a transition between innate PAMP driven responses and the classical MHC restricted response to infection?


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