For the first time in the Veterans and Agent Orange series, immune-system disorders are being addressed in a separate chapter preceding those on other types of adverse health outcomes. In previous Veterans and Agent Orange reports—Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam, hereafter referred to as VAO (IOM, 1994), Veterans and Agent Orange: Update 1996 (IOM, 1996), Update 1998 (IOM, 1999), Update 2000 (IOM, 2001), Update 2002 (IOM, 2003), Update 2004 (IOM, 2005), Update 2006 (IOM, 2007), and Update 2008 (IOM, 2009)—possible adverse health outcomes arising from disruptions of the immune system were included in the Other Health Outcomes chapter. The current committee elected to comprehensively revisit the limited epidemiologic evidence concerning association of immune disease with herbicide exposure in light of the substantial volume of toxicologic evidence of 2,3,7,8-tetrachlorodibenzo-p-dioxin’s (TCDD’s) impairment of the immune systems of laboratory animals. The chapter opens with an overview of the various types of health problems that can arise from malfunctioning of the human immune system. The standard VAO sections leading to the committee’s assignment of a health outcome to a category of association follow and include a new tabulation of all the immunerelated epi-demiologic information that has been considered in this series, plus a synopsis of the information new to this update. The next section discusses a series of factors that may contribute to the immune responses of animals exposed to the chemicals of interest being considerably more pronounced than any observed to date in humans. The chapter closes with the committee’s thoughts for research on the possibility that immune perturbations in humans may function as a mechanistic step in the development of disease processes in other organ systems.
The immune system plays three important roles in the body:
- It defends the body against infection by viruses, bacteria, and other disease-producing microorganisms, known as pathogens.
- It defends against cancer by destroying mutated cells that might otherwise develop into tumors and by providing immunity against tumors.
- It provides resident immune cells that are specially adapted for different tissues and organs (such as microglia in the central nervous system and Kupffer cells in the liver) that help to regulate the functional activity and integrity of those tissues.
To recognize the wide array of pathogens in the environment, the immune system relies on many cell types that operate together to generate immune responses. Those cells arise from stem cells in the bone marrow, they are found in lymphoid tissues throughout the body, and they circulate in the blood as white blood cells (WBCs). The main types of WBCs are granulocytes, monocytes, and lymphocytes. Each category has many specialized cell populations that are responsible for specific functions connected to the production of specific immune hormones (generically known as cytokines). Imbalances in these specialized populations or in their level of functional activity can result in inadequate or improper immune responses that may lead to pathologic outcomes. Diseases arising from immune dysfunction may be apparent immediately or observed only after an organism encounters an environmental challenge that causes immune cells to respond (such as an infection). Immune dysfunctions are in four major categories that need not be mutually exclusive: immune suppression, allergy, autoimmunity, and inflammatory dysfunction (inappropriate and/or misdirected inflammation). Although immune suppression usually is seen as an increased incidence of infections or an increased risk of cancer, allergic, autoimmune, and inflammatory disorders can be manifested as diseases affecting virtually any tissue. It is often difficult to diagnose such diseases, so they may or may not be medically categorized as immune disorders.
Suppression of immune responses can reduce resistance to infectious disease and increase the risk of cancer. Infection with the human immunodeficiency virus (HIV) is a well-recognized example of an acquired immune deficiency in which a specific type of lymphocyte (CD4+ T cells) is the target of the virus. The decline in the number of CD4+ T cells after HIV infection correlates with an increased incidence of infectious diseases, including fatal opportunistic infections, and with an increased incidence of several types of cancer. Treatment of cancer patients with toxic chemotherapeutic drugs suppresses the immune system by inhibiting the generation of new WBCs by the bone marrow and by blocking proliferation
of lymphocytes during an immune response. Both those examples represent severe immune suppression in which the adverse outcome is easily detected with clinical measurements.
Immune suppression can also result from exposure to chemicals in the workplace or in the environment and be manifested as recurrent infections, opportunistic infections, a higher incidence of a specific category of infections, or a higher incidence of cancer. However, unless the immune suppression is severe, it is often difficult to obtain clinical evidence that directly links chemically induced changes in immune function to increased infectious disease or cancer, because many confounding factors can influence a person’s ability to combat infection. Such confounders include age, vaccination status, the virulence of the pathogen, the presence of other diseases (such as diabetes), stress, smoking, and the use of drugs or alcohol. Therefore, immunotoxicology studies are often conducted in laboratory animals to understand the scope and mechanism of chemical-induced immune suppression. Results of such studies can be used to develop biomarkers to assess effects in human populations. Infectious-disease models in animals can also be used to determine whether the pattern of disease changes with chemical exposure.
The immune system sometimes responds to a foreign substance that is not pathogenic. Such immunogenic substances are called allergens. Like most immune-based diseases, allergic diseases have both environmental and genetic risk factors. Their prevalence has increased in many countries in recent decades (CDC, 2004; Linneberg et al., 2000; Simpson et al., 2008; Sly, 1999). Major forms of allergic diseases are asthma, allergic rhinitis, atopic dermatitis, and food allergy. The response to some allergens, such as pollen and bee venom, results in the production of immunoglobulin E (IgE) antibodies. Once produced, IgE antibodies bind to mast cells, specialized cells that occur in tissues throughout the body, including lung airways, the intestinal wall, and blood-vessel walls. When a person is exposed to the allergen again, it binds to the antibodies on the mast cells and caused them to release histamine and leukotrienes, which produce the symptoms associated with an allergic response. Other allergens, such as poison ivy and nickel, activate allergen-specific lymphocytes at the site of contact (usually the skin) that release substances that cause inflammation and tissue damage. Some allergic responses, such as those to food allergens, may involve a combination of allergen-specific lymphocyte–driven and IgE–driven inflammation. Allergic responses may be manifested in specific tissues (such as skin, eye, airways, and gastrointestinal tract) or result in a system-wide response called anaphylaxis.
At least 60 recognized diseases and conditions affecting the cardiovascular, respiratory, nervous, endocrine, dermal, gastrointestinal, hepatic, and excretory systems are classified as autoimmune diseases (WHO, 2006). They affect both men and women. Most of the autoimmune diseases affect more women than men (Fairweather et al., 2008). Genetic predisposition, age, hormone status, and environmental factors, such as infectious diseases and stress, are known to affect the risk of developing autoimmune diseases. The existence of some autoimmune diseases is also a risk factor for the development of other immune-related diseases, such as some types of cancer (Landgren et al., 2010).
Autoimmune disease is an example of the immune system’s causing rather than preventing disease: the immune system attacks the body’s own cells and tissues as though they are foreign. Inappropriate immune responses that result in autoimmune disease can be promoted by different components of the immune system (such as antibodies and lymphocytes) and can be directed against a wide variety of tissues or organs. For example, the autoimmune reaction in multiple sclerosis is directed against the myelin sheath of the nervous system; in Crohn disease, the intestine is the target of attack; in type 1 diabetes mellitus, the insulin-producing cells of the pancreas are destroyed by the immune response; rheumatoid arthritis arises from immune attack on the joints.
More generalized forms of autoimmune diseases also occur. Systemic lupus erythematosus (SLE) is an autoimmune disease that has no specific target organ of immune attack. Instead, patients have a variety of symptoms that often occur in other diseases, and this makes diagnosis difficult. A characteristic rash across the cheeks and nose and sensitivity to sunlight are common symptoms; oral ulcers, arthritis, pleurisy, proteinuria, and neurologic disorders may be present. Almost all people who have SLE test positive for antinuclear antibodies in the absence of drugs known to induce them. The causes of SLE are unknown, but environmental and genetic factors have been implicated. Some of the environmental factors that may trigger it are infections, antibiotics (especially those in the sulfa and penicillin groups) and some other drugs, ultraviolet radiation, extreme stress, and hormones. Occupational exposures to such chemicals as crystalline silica, solvents, and pesticides have also been associated with SLE (Cooper and Parks, 2004; Parks and Cooper, 2005).
Inflammatory diseases make up a more recently identified category of immune-related disorders characterized by dysfunctional inflammatory responses (usually involving immune cells) that are exaggerated, excessively prolonged, or misdirected. Tissue disease can result from this inappropriate inflammation,
which can affect virtually any organ. Examples of diseases and other conditions that are most often included in other disease categories but are also considered to be inflammatory diseases are coronary arterial disease, asthma, eczema, chronic sinusitis, hepatic steatosis, psoriasis, celiac disease, and prostatitis. Inflammatory diseases often occur with one another, and this has resulted in the categorizing of different but linked inflammatory diseases together as a single chronic inflammatory disorder (Borensztajn et al., 2011); among these are atherosclerosis and chronic pulmonary obstructive disease. Inappropriate inflammation also appears to play a role in promoting the growth of cancer (Bornschein et al., 2010; Hillegass et al., 2010; Landgren et al., 2010; Porta et al., 2010; Winans et al., 2010). Examples of this can be seen in the higher prevalence of specific cancers in patients who have such inflammatory diseases as inflammatory bowel disease (Lucas et al., 2010; Viennot et al., 2009; Westbrook et al., 2010), prostatitis (Sandhu, 2008; Wang et al., 2009) and psoriasis (Ji et al., 2009).
Ordinarily, inflammation can be advantageous in fighting infectious diseases. It is one component of the normal host response to infection and is mediated by innate immune cells. Inflammatory responses have evolved to speed the trafficking of macrophages, granulocytes, and some lymphocytes to the area of infection, where they produce toxic metabolites that kill pathogens. Interactions among innate immune cells and epithelial and endothelial cells are important in regulating the level of inflammation. However, improperly regulated inflammation can contribute to diseases that arise in nonlymphoid tissues such as the lungs, skin, nervous system, endocrine system, and reproductive system.
The following comments are restricted to findings on the immune system after adult human exposure. For a discussion of potential effects on the immune system arising from early-life (such as perinatal) exposures (which would not be directly applicable to the Vietnam veterans who are the target of this report), see Chapters 4 and 8. Studies that served as the basis of prior updates of VAO and one 2009 study are shown in Table 6-1.
A handful of the direct studies of veterans listed in Table 6-1 reported a statistical difference in a single immune measure (Kim et al., 2003; Michalek et al., 1999a). But invariably the same effect was not found in other studies of Vietnam veterans, nor was support found in epidemiologic studies of other populations. Thus, there were no consistent findings indicative of immunosuppression, increased risk of autoimmunity (usually as measured with autoantibodies), or biomarkers of atopy or allergy (such as increased IgE concentrations). Much of
|US Air Force Health Study (AFHS)—Ranch Hand veterans vs SEA veterans||All COIs|
|AFHS, 2000||Participants in 1987 examination cycle, Ranch Hands vs comparisons—mortality||A small dose-related increase in T-cell counts and a high-dose increase in NK markers, neither considered by authors to be biologically important; no dose–response relationship for TCCD exposure associated with T-cell activation markers (CD25), serum Ig, or autoantibodies|
|Michalek et al., 1999a||Participants in 1997 examination cycle, Ranch Hands vs comparisons—incidence||No change in surface markers for B and T cells, no change in serum Ig, no change in autoantibodies (antinuclear antibody, smooth muscle autoantibody, parietal cell autoantibody, rheumatoid factor, and monoclonal immunoglobulins) and no dose-related change in DTH response|
|Wolfe et al., 1990||Participants in 1987 examination cycle, Ranch Hands vs comparisons—morbidity||No change in surface markers for B and T cells|
|Wolfe et al., 1985||Participants in 1985 examination cycle, Ranch Hands vs comparisons— morbidity and mortality||No change in surface markers for B and T cells|
|US CDC Vietnam Experience Study (VES)||All COIs|
|Boehmer et al., 2004||Mortality (1965–2000)||No increase in infectious or parasitic diseases|
|CDC, 1988b||Deployed vs nondeployed—morbidity||No differences in infections, no changes in B and T cell-surface markers, WBC counts, or circulating serum Ig|
|US VA Cohort of Monozygotic Twins||All COIs|
|Eisen et al., 1991||Physical health—morbidity||Increase in skin conditions of unknown etiology, no increase in blood disorders|
|American Legion Cohort||All COIs|
|Stellman et al., 1988||Physical health and reproductive outcomes||Increase in skin conditions and arthritis|
|State Studies of US Vietnam Veterans||All COIs|
|Visintainer et al., 1995||Michigan Vietnam Veterans (deployed vs nondeployed)||Increased mortality from infectious (including parasitic) diseases|
|Kahn et al., 1992||New Jersey Agent Orange Commission||Depressed response to tetanus in DTH tests, decrease in CD4 and SmIg+ B cells|
|Newell, 1984||Agent Orange Advisory Committee of Texas||Increase in percentage of active T rosette-forming cells|
|Australian Vietnam Veterans||All COIs|
|O’Toole et al., 2009||Australian Vietnam Veterans—longitudinal cohort study of 67 conditions in randomly selected Vietnam veterans vs general population||Increase in hay fever, increases in infectious and parasitic diseases, increase in arthritis|
|CDVA, 1997b||National Service Vietnam Veterans—mortality||No change in mortality from infectious (including parasitic) diseases|
|Korean Vietnam Veterans||All COIs|
|Kim et al., 2003||Immunotoxicologic study||Increase in IgE and IL-4, decrease in IgG1 and IFN-gamma, no change in lymphocyte counts|
|Vietnamese Vietnam Veterans||All COIs|
|Chinh et al., 1996||Antinuclear and sperm autoantibodies||No change in autoantibodies to sperm, antinuclear bodies|
|Chemical or Industrial Workers|
|Baranska et al., 2008||A prospective multicenter cohort study of 238 pesticide-exposed workers vs 138 unexposed workers||Pesticide factories (not specifically TCDD): Reduced antibody responses to hepatitis B vaccination among exposed workers carrying a specific IL-1 allele|
|Neubert et al., 2000||Updated and expanded evaluation of 158 workers in a German chemical plant with differing exposure studied in two trials||TCDD (or “TCDD toxic equivalents” from PCDD/PCDF): No differences in serum Ig or cytokine (IL1, IL6, TNF-alpha)|
|Ernst et al., 1998||19 highly exposed chemical workers vs 28 unexposed controls in two chemical plants in Hamburg, Germany||TCDD (in chemical plant): In subset of leukocytes, increase in CD8+ memory T cells and decrease in naïve T cells (CD45RA+) after TCDD exposure, as was stimulated IFN-gamma production from whole blood cultures associated with TCDD exposure|
|Halperin et al., 1998||Cross-sectional study of 259 TCDD-exposed 2,4,5-trichlorophenate (and its derivatives) workers (mean serum TCDD, 223 ppt) and 243 unexposed residential controls (mean serum TCDD, 6 ppt)||TCDD (exposure in a chemical plant): No significant changes in serum Ig or major leukocyte categories; TCDD associated with decreased circulating CD26 cells (activated T cells)|
|Jung et al., 1998||192 workers in a German pesticide plant, including 29 highly exposed and 28 controls compared for immune functional tests||TCDD (or TEQs from PCDD/PCDF exposure): No signifcant changes in TCDD and lymphocyte subsets, antibody responses to vaccination, lymphocyte proliferation, or autoantibody production; decrease in chromate resistance of PHA-stimulated lymphocytes in highest exposure group|
|Sweeney et al., 1997/1998||1987 cross-sectional study of 281 chemical-plant workers in NJ and MO at least 15 years after exposure vs 260 unexposed controls||TCDD (as a contaminant in chemical production): Increase in TCDD associated with a decrease in CD3/Ta1 (helper lymphocytes) cells|
|Tonn et al., 1996||Comparison of 11 2,4,5-trichlorophenol production workers 20 years after exposure vs 10 unexposed age-matched workers in the same company||TCDD: No differences in any lymphoid subset or in mitogen-induced proliferation; TCDD exposure was associated with decreases in MLR response and in stimulation with IL-2 in vitro|
|Jansing and Korff, 1994||Examination of eight trichlorophenol production workers who developed chloracne and were re-examined 15–25 years after initial exposure||TCDD: Reduced gamma globulins in the most-exposed workers; no significant effects on T4, T8 ratios|
|Benner et al., 1994||Cross-sectional study of 153 male workers in six chemical plants in Germany||TCDD (during production of TCP): DTH responses not correlated with dioxin concentration; slight decrease in IgM was reported with increasing dioxin exposure; overall lymphoid counts not different|
|Ott et al., 1994||138 surviving workers from a larger cohort of 254 exposed workers after an accident in a BASF TCP production facility||TCDD: Among 14 immune measures; regression analysis of TCDD concentration suggested marginal positive associations with IgG, IgA, C3, and C4; marginal reductions in some lymphocyte population were also reported|
|Neubert et al., 1993, 1994||89 volunteers involved in decontamination work at a chemical plant in Hamburg, German; no control population||TCDD (or equivalents via PCDD/PCDF exposure): Potentially complicated by age differences among the compared groups; only subtle, clinically nonsignificant changes were seen among immune-cell surface markers in a comparison of higher exposed vs low-exposed to moderately exposed workers|
|Jennings et al., 1988||18 chemical workers in a 2,4,5-T factory exposed as a result of an industrial accident 17 years before study vs 15 matched controls||TCDD: No changes in serum Ig classes, increases in antinuclear antibodies and immune complexes, and increase in circulating NK cells (Leu7+) in exposed workers|
|Waste Incinerator Workers|
|Oh et al., 2005||Comparison of immune measures in 31 waste- incineration workers vs 84 controls||TCDD (via waste incineration): Lymphoid subsets, IFN-gamma, and Ig not statistically different; decrease in IL-4 and increase in T-cell activation (measured as combined CD3 and CD69 markers) associated with TCDD exposure|
|Agricultural Health Study (AHS)||Various categories of agricultural pesticides|
|Beseler et al., 2008||Comparison from the AHS of 534 cases of self-reported physician- diagnosed depression vs 17,051 controls||Both high-level acute pesticide exposure (OR = 2.57, 95% CI 1.74–3.79) and cumulative pesticide exposure (OR = 1.54, 95% CI 1.16–2.04) were positively associated with increase in depression|
|Beseler et al., 2006||29,074 female spouses of pesticide applicators in the AHS||Depression was significantly associated with pesticide poisoning (OR = 3.26, 95% CI 1.72–6.19) but not with lower cumulative exposure|
|De Roos et al., 2005b||Nested case–control study of rheumatoid arthritis in agricultural families (57,000 pesticide applicators and their spouses).||No strong risk factors were identified for pesticide mixing or application or for any specific class of pesticides in the AHS of rheumatoid arthritis.|
|Other Agricultural Studies|
|Faustini et al., 1996||Longitudinal study of 10 farmers during 1994 within 7 days before and 1–12 days and 50–70 days after exposure||2,4-D and MCPA formulations: Decreases in percentages of CD4, CD8, CTL, CD8-DR, and NK cells and in NK activity and mitogen-stimulated lymphoproliferation; CD4:CD8 ratio was unaltered; CD3 and CD8 percentages had recovered by the second assessment period; no significant correlations between immune changes and amount of pesticides applied|
|Seveso Cleanup Workers||TCDD|
|Ghezzi et al., 1982||Prospective study using analysis of samples from 36 cleanup workers (divided into three groups based on time spent in the contamination area); pre-employment samples and samples after 9 months were analyzed for comparison with samples from 31 nonexposed workers||No differences in WBC counts and platelet counts|
|Seveso Residential Population||TCDD|
|Baccarelli et al., 2005b||Study of 101 chloracne cases vs 211 controls 20 years after the accident; relatively low statistical power was available because the study examined the occurrence of individual diseases||Persistent increase in TCDD in chloracne cases; younger people seemed to be more susceptible; no major trends in disease occurrence|
|Baccarelli et al., 2002||Study of 62 people from a highly exposed zone and 53 from noncontaminated areas 20 years after the accident Plasma concentration of TCDD was determined; multivariate regression analysis showed significant decrease in plasma IgG with increasing TCDD concentration and no changes in IgM, IgA, or C3|
|Pocchiari et al., 1979||45 children (3–7 yrs of age) living in exposed areas vs 45 nonexposed children as controls||No differences in serum IG, mitogen responses of lymphocytes (PHA and pokeweed), or percentage of rosette- forming lymphocytes|
|Times Beach (MO) Cohort||TCDD|
|Webb et al., 1987||82 people in more highly contaminated areas vs 40 in low-risk exposure areas as controls||No differences in DTH response or T-cell subsets (T4/T8)|
|Stehr et al., 1986||80 people in highly contaminated areas vs 40 controls in lower-risk areas||No differences in DTH induration or T-cell subset analysis (T4/T8)|
|Knutsen, 1984||Pilot study of small numbers of people; for comparisons, people were assigned to two environmental-exposure groups: those in high-risk areas (27 men, 23 women, and 15 children) and those in low-risk areas (12 men, 10 women, and 8 children)||Multitest DTH evaluation to seven recall antigens was performed, no statistical differences were reported, and only trends were noted; no statistical differences were reported for T-cell markers (T3, T4, and T8) or mitogen-induced lymphocyte proliferation (PHA, Con A, and pokeweed mitogen), and only trends were noted|
|Quail Run Mobile Home Park (MO) Cohort||TCDD|
|Evans et al., 1988||A subset of the previously anergic persons in the Stehr-Green et al. (1987) study were re-evaluated in the DTH test with a higher DTH test dose and highly trained, blinded readers||Retesting of DTH failed to produce the differences observed initially|
|Knutsen et al., 1987||Small (ill-defined) samples were used; comparisons of residents of the Quail Run Mobile Home Park with residents of St. Louis–area trailer parks as controls||DTH suppression in the exposed group was reported, but data from two of four readers were discarded; no differences in T-cell mitogen stimulation; decreases in percentages of T3, T4, and T11 cells in the exposed group|
|Stehr-Green et al., 1987||154 people in highly contaminated area vs 155 in three low–environmental- contamination areas as controls||Increase in anergy and decrease in induration for DTH in exposed group; data from some readers were excluded; decrease in percentages of T3, T4, and T11 cells, but no difference in cell number of T4/T8 ratio.|
|Andrews et al., 1986||80 people in a high– exposure risk group vs 40 controls||Decreases in DTH indurations, number of positive reactors, and percentages of T3, T4, and T11 cells in the exposed group|
|Hoffman et al., 1986||154 people in the exposed area vs 155 non-exposed people in an uncontaminated area||Recall antigen multitest for DTH, increase in percentage of anergy and decrease in induration in exposed group; data from two of four readers were excluded|
|Missouri Residential Population||TCDD|
|Webb et al., 1989||Regression analysis used for comparisons among 41 exposed people for adipose- tissue TCDD vs immune measures; three exposed groups defined by tissue dioxin||No TCDD–DTH response relationships were reported; no change in mitogen responsiveness; some serum markers (A/G ratio and serum IgG) were affected|
|Other Environmental Studies|
|Lee et al., 2007a||NHANES—1,721 adults were assessed for serum dioxin-like PCBs and self-reported arthritis||Dioxin-like PCBs: Association between serum dioxin-like PCBs and prevalence of arthritis particularly among women|
|Van den Heuvel et al., 2002||200 people 17–18 yrs of age in three areas of Flanders (Belgium); TEQ values were calculated from serum dioxin-like PCB concentrations and relationships with immune measures were examined||Dioxins and PCBs: Decreases in eosinophil and NK-cell counts with increasing TEQ; IgE concentrations; history of upper airway allergy, and odds of a positive RAST test correlated negatively with serum TEQ; IgA concentrations correlated positively with TEQ|
|Lovik et al., 1996||Blood samples from 24 Norwegian hobby fishermen were compared with those of 10 male referents as controls||PCDD, exposure from food: The study generally lacks experimental details; no differences in an NK cell marker or in NK activity were seen; apparently, some effects on lymphoid markers were observed but specific details are lacking|
|Wolf and Karmaus, 1995||Cross-sectional study of 221 teachers who worked in German day-care centers treated with wood preservatives vs 189 teachers who worked in untreated facilities||Dioxin in wood preservatives, exposure primarily via inhalation: No effects of inhaled dioxin were seen on T4 or T8 cell numbers or on the ratio; some evidence of a dose–response relationship was seen for risk of anergy (or hypoergy) in the DTH assay|
|Svensson et al., 1994||23 high consumers of fatty fish from the Baltic Sea (containing low concentrations of PCDD) vs 20 low consumers or nonconsumers of fish as controls||PCDD, exposure from food: Blood PCDDs were significantly different between the groups; mercury concentrations also differed; NK cells correlated negatively with blood concentrations of persistent organic chemicals; no other differences were found in lymphocyte populations or in mitogen stimulation of lymphocytes|
|Hardell et al., 1987||Telephone interviews concerning environmental and occupational chemical exposures were conducted with 50 AIDS patients (with Kaposi sarcoma) and 50 homosexual men as controls||Chemical exposures, including pesticides, and Agent Orange: No significant differences were reported in a small study that generally lacked focus|
|ABBREVIATIONS: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; AFHS, Air Force Health Study; AHS, Agricultural Health Study; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemical of interest; Con A, concanavalin A; DTH, delayed-type hypersensitivity; IFN-gamma, interferon-gamma; Ig, immunoglobulin; IL, interleukin; MCPA, methyl-4-chlorophenoxyacetic acid; MLR, mixed lymphocyte response; NHANES, National Health and Nutrition Examination Survey; NK, natural killer; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzo-p-dioxin (highly chlorinated, if four or more chlorines); PCDF, polychlorinated dibenzofurans; PHA, polyhydroxyalkanoates; RAST, radioallergosorbent; SEA, Southeast Asia; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCP, trichlorophenol; TEQ, total toxic equivalent; TNF, tumor necrosis factor; VA, Department of Veterans Affairs; VES, Vietnam Experience Study; WBC, white blood cell.|
the focus of the studies was on measuring T4/T8 ratios. The T4/T8 ratio is an effective biomarker of the progression of HIV-induced AIDS, but, on the basis of the TCDD-exposure animal data, it is not an immunologic index that is expected to be altered.
Occupational-exposure studies shown in Table 6-1 evaluated concentrations of lymphoid populations in circulation, such as CD4, CD8 (and their ratio), and natural killer (NK) cells; cell-mediated immunity (the delayed-hypersensitivity response); serum concentrations of immunoglobulins, such as IgM, IgG, and IgA; concentrations of complement, such as C3 and C4; and concentrations of cytokines, such as IL-1, IL-2, interferon-gamma, IL-4, IL-6, and tumor necrosis factor-alpha. A few studies also included disease or condition end points, such as
rheumatoid arthritis, SLE, and depression. Ex vivo analyses included measures of NK activity, lymphoid mitogen-induced proliferation, and the mixed lymphocyte response (MLR) against allogeneic cells. Some studies identified one or more dioxin-related shifts in immune measures, but many reported no significant differences in the same measures. That is particularly true of the study by Neubert et al. (2000), which measured toxicity equivalents (TEQs) for dioxin but found no immunoglobulin or cytokine alterations. In general, the spectrum of occupational-exposure findings does not provide a consistent or clear picture of alterations in immune measures that could be extrapolated to an increased risk of a single disease or even a broader category of diseases. The exception may be observations of pesticide-associated autoimmunity and depression. Immune depression was rather consistently associated with very high pesticide exposures or pesticide poisonings. However, because the studies generally concerned broad categories of pesticide exposure, their relevance to herbicide exposures in Vietnam is not clear.
Several environmental-exposure studies reported alterations, but findings were inconsistent among the studies (Table 6-1). Some studies reported alterations in immune measures associated with TEQs for dioxin. For example, Van den Heuvel et al. (2002) reported that IgE, positive radioallergosorbent (RAST) tests in response to specific allergens, eosinophils counts, and NK cell counts correlated negatively with dioxin TEQs, but that IgA increased; these alterations, however, were not seen consistently in other studies. Baccarelli et al. (2002) found no changes in IgA but saw changes in IgG in the Seveso population. Svennson et al. (1994) found that NK cell numbers were reduced with increasing concentrations of persistent organic chemicals, but Lovik et al. (1996) found no difference in NK numbers or activity. Similarly, the occupational-exposure studies (Table 6-1) that examined NK concentrations reported the full spectrum of results: no alterations (Halperin et al., 1998), a decrease (Faustini et al., 1996), and even an increase in NK numbers (Jennings et al., 1988) in dioxin-exposed people.
As seen Table 6-1, some early studies of the Quail Run Mobile Home Park population exposures reported that dioxin exposure was associated with a reduced cell-mediated immune response, the delayed-type hypersensitivity (DTH) response (Andrews et al., 1986; Hoffman et al., 1986; Knutsen et al., 1987; Stehr-Green et al., 1987). But some of those studies had technical problems in assessment and in followup analyses. Dioxin-associated changes were not confirmed (Evans et al., 1988; Webb et al., 1989). In addition, several studies of the Times Beach population did not find any alteration of the DTH response in dioxin-exposed populations (Knutsen, 1984; Stehr et al., 1986; Webb et al., 1987).
Analysis of National Health and Nutrition Examination Survey (NHANES) data found that exposure to dioxin-like PCBs was associated with an increase in
self-reported arthritis (Lee et al., 2007a), but De Roos et al. (2005b) had found no such association in their study.
Prior VAO updates concluded that human data were either insufficient or inconsistent with respect to an increased risk of immunosuppression, allergic disease, or autoimmune disease.
For this update, the committee revisited the entire literature of herbicide– human immune findings from studies of Vietnam veterans, occupationally exposed people, and environmentally exposed people (Table 6-1), including studies reviewed in prior VAO updates and one study published since Update 2008.
Among the previously considered human studies, only two stand out for special consideration on the basis of their analysis of actual immune-based disease or clinically relevant human immune responses. Zober et al. (1994) studied three categories of occupationally exposed workers based on chloracne status (chloracne not evident, moderate, or severe) and nonexposed workers. They found that the frequencies of episodes of parasitic diseases, respiratory infections, and skin diseases were elevated with respect to the nonexposed workers (p = 0.067, p = 0.003, and p = 0.001, respectively), and each of these outcomes showed increasing trends over the three chloracne categories (an indicator of higher dioxin exposure). Baccarrelli et al. (2002) reported that higher TCDD exposure was associated with lower serum IgG in the exposed Seveso populations.
Only one new epidemiologic study addressed exposure to the chemicals of interest and outcomes in which immune function may play a prominent role. Infectious and parasitic diseases, respiratory disorders, and skin disorders were among the many conditions that O’Toole et al. (2009) found to be significantly more prevalent in Australian Vietnam veterans, on the basis of self-reports, than in the general population. The confidence that can be placed in this new study is substantially hampered by a poor response rate, its reliance on self-reported diagnoses, the questionable suitability of the general population as a control group, and the fact that the veterans and the controls were interviewed under quite different circumstances. Reporting bias and a “healthy-warrior” effect might be expected to bias the findings in opposite directions, but the near uniformity of significant findings on these self-reported health problems in the deployed veterans suggests that problems associated with reporting bias may have been dominant.
In combination, the studies raise the question of whether high TCDD exposure may contribute to a reduced ability to fend off or to clear some types of infections.
There is an extensive body of evidence from experimental studies in animal-model systems that TCDD, other dioxins, and several dioxin-like chemicals (DLCs) are immunotoxic (Kerkvliet, 2009). Immunotoxicity is due primarily to changes in adaptive immune responses that result in suppression of both antibody and cell-mediated immunity and a reduction in the ability to clear pathogenic infections and prevent tumor growth. Studies in laboratory mice have shown that the immunotoxicity of TCDD and DLCs depends on activation of the arylhydrocarbon receptor (AHR). Most of the cell types involved in the immune system express the AHR, so there are many potential pathways to im-munotoxicity. TCDD has also been shown to alter macrophages and neutrophils in a manner that exacerbates some forms of inflammation during infections and may contribute to the development of chronic inflammatory lung disease (Teske et al., 2005; Wong et al., 2010).
TCDD is a potent immunosuppressive chemical in laboratory animals. The relative potencies of given DLCs based on induction hepatic enzymes (their toxicity equivalency factors [TEFs]) appear to predict the degree of immunosuppression induced (Smialowicz et al., 2008). Exposure of animals to dioxin not only suppresses some adaptive immune responses but also has been shown to increase the incidence and severity of various infectious diseases and to increase the development of cancer (Choi et al., 2003; Head and Lawrence, 2009; Jin et al., 2010). It is consistent with its immunosuppressive effects that TCDD exposure suppresses the allergic immune response of rodents, and this in turn results in decreased allergen-associated pathologic lung conditions and has recently been shown to suppress the development of experimental autoimmune disease (Quintana et al., 2008). Thus, depending on the disease, TCDD exposure could result in exacerbation or amelioration of symptoms.
Recent attention has focused on the ability of the AHR to induce regulatory T cells (Marshall and Kerkvliet, 2010). These so-called Tregs have potent suppressive activity in the immune system, and their inappropriate induction by TCDD could account for much of the immune suppression. AHR activation in dendritic cells has also been shown to promote the development of Tregs by inducing tryptophan metabolism. AHR activation in B cells can directly disrupt the production of antibodies (Sulentic and Kaminski, 2011). The recent demonstration that AHR activation by TCDD leads to the development of regulatory T cells helps to explain the diversity of effects seen after exposure to TCDD (Funatake et al., 2008; Marshall et al., 2008; Quintana et al., 2008).
One would expect exposure to substantial doses of TCDD to result in immune suppression in Vietnam veterans. However, several studies of various measures of human immune function failed to reveal consistent correlations with TCDD exposure, probably because the exposures were inadequate to produce immune suppression or the characteristics measured were not among those most relevant with respect to biological plausibility. No clear pattern of an increase in infectious disease has been documented in the studies of veterans exposed to TCDD or to the herbicides used in Vietnam. However, one occupational-exposure study and one environmental-exposure study do support the possibility that sufficiently high exposure to TCDD may result in an increased frequency of infections. It was also supported by the self-reporting study by O’Toole et al. (2009). As a result, frequency and duration of specific types of infections should be a focus of future studies. Suppression of the immune response by TCDD might increase the risk of some kinds of cancer in Vietnam veterans, but there is no evidence to support that connection.
Allergic and Autoimmune Diseases
Epidemiologic studies have been inconsistent with regard to TCDD’s influence on IgE production in humans. No human studies have specifically addressed the influence of TCDD on autoimmune disease, but several animal studies have shown that TCDD suppresses the development of autoimmune diseases. In studying postservice mortality, Boehmer et al. (2004) found no increase in deaths of Vietnam veterans that could be attributed to immune-system disorders. The present committee’s review included a study that found a significant association between concentrations of dioxin-like PCBs and the prevalence of arthritis in women but not in men (Lee et al., 2007a). There is no experimental evidence to support that finding, but increased inflammatory responses could be involved. Future studies are needed to determine a potential mechanism of TCDD-induced rheumatoid arthritis.
Few effects of phenoxy herbicide or cacodylic acid exposure on the immune system have been reported in animals or humans, and no clear association between such exposure and autoimmune or allergic disease has been found. Exposure of laboratory animals to phenoxy herbicides or cacodylic acid has not been associated with immunotoxicity.
There are no human data on the potential for dioxin or the herbicides of interest to induce dysregulation of inflammation that could contribute to an increased risk of inflammation-associated diseases.
Possible associations involving infectious or inflammation-related diseases should be a focus for the future. Examples of studies that would add support for these potential adverse outcomes are Baccarelli et al. (2002), Baranska et al. (2008), Beseler et al. (2008), Oh et al. (2005), O’Toole et al. (2009), Tonn et al. (1996), and Visintainer et al. (1995).
On the basis of the evidence reviewed here and in previous VAO reports, the present committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the chemicals of interest and specific infectious, allergic, or autoimmune diseases.
Animal studies and in vitro studies with human cells and cell lines are important ways of trying to understand underlying biologic mechanisms associated with immunotoxic and other responses to xenobiotics, which are “foreign” substances that do not normally occur in biologic systems. However, as discussed above, despite the vast array of data supporting the immunotoxicity of TCDD in laboratory animals, there is little evidence from studies of Vietnam veterans or other human populations that suggests that TCDD or the herbicides of concern produce immune alterations. Many factors must be considered in examining the relevance of animal and in vitro studies to human disease and disease progression, and they are discussed Chapter 4. Here, we present the factors that are probably most important in considering differences between the results of laboratory studies and the findings of observational epidemiologic studies.
Magnitude and Timing of Exposure
In general, the TCDD exposures used in animal studies have been orders of magnitude higher than Vietnam veterans are likely to have received during military service. It is well known that the immune system is highly susceptible to xenobiotic exposure during critical stages of development, such as gestation. It is also well known that primary immune responses are easier to alter than secondary immune responses. In vivo studies show that exposure to antigens may be important, so the timing of antigen exposure relative to TCDD exposure may be an important variable.
Human immune diseases are likely to have complex etiologies and to be under the influence of numerous genes and gene-by-environment interactions (Dietert et al., 2010). Differences in AHR affinity between species may be a factor in animal-to-human extrapolation. For example, many strains of mice (AHRb) are known to exhibit greater susceptibility of CYP1A1 induction and immune suppression than other strains (AHRd). In contrast, a simple single-haplotype difference in susceptibility to TCDD has not been observed in humans. Rats appear to be more similar to the resistant AHRd phenotype of mice in their sensitivity to TCDD. Indeed, it is difficult to produce immune suppression in rats with TCDD because of that, and there probably are other genetic reasons as well.
There are well-known differences in the susceptibility to xenobiotic exposures between male and female animals. There are probably multiple reasons for the differences, some of which may pertain to immunomodulation by sex steroids. Similarly, evidence suggests that specific immune-based health risks in humans have important sex differences. For example, women generally are much more susceptible to the development of several autoimmune diseases than men; such differences in humans may result from a combination of genetic factors and environmental exposures. That has ramifications for future studies. In considering the potential impact of Agent Orange on the immune system and the risk of disease, sex-based differences in chemically induced adverse immune outcomes need to be investigated. Future studies should ensure that, whether in animal models or in direct human studies, gene- or sex-specific immune effects are able to be evaluated with sufficient statistical power to support distinctions.
Stress produced is a well known modifier of human immune responses. It is an ever-present variable that is difficult to assess or control for in epidemiologic studies.
Immune biomarkers (such as cytokines, antibodies, antitumor activity, populations of specialized cells, and inflammatory metabolites) can be used to examine the risks of such specific health problems as heightened allergic responses, deficiency in cell-mediated immunity, and susceptibility to autoimmune responses. In addition, there may be more generalized biomarkers; for example, a recent human study reported that the concentrations of a specific cytokine produced by macro-
phages (macrophage inhibitory cytokine-1) was a useful biomarker for predicting all-causes mortality (in subjects who already had particular chronic diseases) over a span of 14 years (Wiklund et al., 2010). In the absence of clearly defined immune diseases, combinations of immune measures may be used as biomarkers of altered immune responses associated with risks of specific diseases. As a result, antibody concentrations, recall antigen tests, lymphoid subpopulation sizes, and cytokine, receptor, and metabolite concentrations are often used in combination for the prediction of immune-associated health risks. Immune biomarkers, when appropriately selected, could provide useful information regarding potential immune-associated health risk connected with TCDD. However, it is critical that the biomarkers used in such studies be those most predictive for risk of disease, and not just those most readily measured.
On the basis of extensive animal studies involving TCDD, the most plausible immune alterations expected in dioxin-exposed human adults are suppression of selected adaptive immune responses and misregulated inflammation. Several human studies (Baccarelli et al., 2002; Halperin et al., 1998; Jung et al., 1998; Michalek et al., 1999a) have examined measures that could reflect functional immune suppression (for example the DTH recall antigen test and concentrations of various antibodies). However, most studies have failed to show a significant effect of dioxin exposure on those measures. Regulation of inflammation is best assessed under the conditions of vaccination or infectious challenge rather than in a resting state. Biomarkers of inflammation would normally include the cyto-kines TNF-a, TGF-b, IL-6, IL-8, IL-10; receptors for TNF-a and IL-6, VCAM-1, ICAM-1, PGE2 and thromboxane; and C-reactive protein–reactive oxygen species production and nitric oxide production. Although a handful of studies included resting (unchallenged) measures for one or two of those biomarkers, no comprehensive testing or challenge-associated analysis has been performed. That constitutes a data gap. Finally, additional studies should focus on novel immune subpopulations, such as Fox p3+ T regulatory cells, Th17 cells, and dendritic cells, on which dioxin has reportedly exerted effects in laboratory animals (Chmill et al., 2010; Jin et al., 2010; Marshall and Kerkvliet, 2010).
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