Immunological Effects of Tobacco Smoking in “Healthy” Smokers

Abstract

The history of tobacco smoking is quite extensive having existed for many years. Over the past 10–15 years significant gains have been made in curbing exposure to tobacco smoke, but tobacco smoke continues to contribute to significant disease. The immunological effects of cigarette smoking have been evaluated and can provide important insight into mechanism of disease. With the extensive growth in elucidation of basic immune processes within healthy host, further research is possible to aide understanding of immune alterations associated with smoking. Specifically, in the area of cellular immunity in which over 30 different cytokines contributing various regulatory functions in immunity, there are many proteins and mechanisms that may be altered. This review will cover the current state of knowledge in the areas of humoral, cellular immunity including results of cytokine studies in healthy smokers.

Key words: :

• Tobacco
• Antibody
• Cytokines
• Interleukin

INTRODUCTION

History of smoking

The Solanaceae family is a large, diverse and economically important family of shrubs and herbs comprising 94 genera and over 3,000 species. The Solanaceae family includes plants such as belladonna, the potato, tomato, eggplant, peppers, petunias, and the tobacco plant. Tobacco originated in the subtropical regions of North and South America. Best estimates place the origins of the tobacco plant around 6000 BCE and the use of tobacco smoke took place in 1 BCE by inhabitants of the Americas ([1]).

Tobacco smoking is thought to have evolved from the use of smoke during religious ceremonies by priest for various effects including healing and therapeutic benefits. By the time of the arrival of the Spanish in Central America, the Mayan and Aztecs were smoking tobacco in a pipe for its pleasure or intoxicating effects. There are no findings to suggest Greek or Roman use of tobacco for smoking as it is currently known. The origin of tobacco in sub-tropical areas further supports the origin of tobacco smoking in the American continents. This includes Canada where exploration found evidence of Indians who smoked ([1]).

By the early 1500s European explorers introduced smoking to Europe, and initiated cultivation of the plant. Tobacco became a lucrative crop based on increasing use in pipe and cigar smoking, as well as chewing tobacco and the use of snuff. A major advancement for the tobacco industry was in the discovery of the cigarette around 1865, but it was not until World War I that cigarette smoking rapidly grew ([2]). Cigarette smoking reached peaked levels with improvements in curing tobacco, increased production and increased availability of cigarettes during the early 1900s. WW I veterans were provided free cigarettes and had one of the highest rates of tobacco use. Interested readers are encouraged to read the books by Corti and Lock for further history on tobacco smoking.

Over the recent years cigarette use in the United States (US) has decreased. In 2000, approximately 23.3% of adults in the US or 46.5 million adults were current smokers (range 13–30%) compared to 25% in 1993. The prevalence of smoking is highest among persons 18–24 years and 25–44 years of age ([3]). The prevalence of tobacco smoking was estimated at 22.5% in 2002 with successive decreases to 21.6% and 20.9% in 2003 and 2004, respectively. Approximately one-half of adult males smoked in the mid-1960s, compared to less than a third of men in a 2005 report ([4]).

Over 400,000 people die in the United States from cigarette smoking per year ([5]). One in five deaths in the United States is smoking-related. Each year 278,000 men and 152,000 women die from smoking. Men who smoke increase their risk of lung cancer 22 times, and increase the risk of bronchitis and emphysema by nearly 10 times. Women who smoke increase their risk of dying from lung cancer by 12 times and they have more than a 10-fold increased risk of dying from bronchitis and emphysema as compared to non-smoking women.

The effects of cigarette smoking on heart disease are well documented. Cigarette smokers experience a 1.5-to 3-fold or greater risk for coronary heart disease (CHD) than do nonsmokers ([6], [7]). This increased risk is independent of other major risk factors for CHD. Each factor contributes about the same order of magnitude of risk for CHD. When one factor is present, the risk approximately doubles, with two factors the risk is fourfold greater; and when all three are present, the CHD risk is eightfold greater than when none of the three factors are present. The risk of CHD is dose-related when measured by the number of cigarettes smoked per day, and decreases with cessation of smoking ([8]).

Smoking triples the risk of dying from heart disease in middle-age men and women, and may contribute to the inflammatory process seen in atherosclerotic cardiovascular disease. Prospective mortality studies involving over 20 million person-years show a 70% greater CHD death rate in smokers compared to non-smokers. Heavy smokers (two or more pack/day) have almost 200% CHD mortality rate compared to non-smokers ([9]). As well as the risk of cancer and CHD associated with smoking, there are increased risks of other disorders including increased infections and asthma ([14]).

It is well recognized that tobacco smoking causes significant disease and mortality. Therefore, it is very important to understand the pathogenesis associated with tobacco smoking, with particular emphasis on the immune pathogenesis given the large occurrences of infections and neoplasia in smokers.

Immunology of smoking

The immune system is an intricate network of various components and signals executed through various cells, proteins, and genetic signals. The signals contribute to amplification of the response to invaders and regulatory control of the overall immune reactions leading to neutralization and eradiation of foreign agents (toxin or biologic). Over the years details of the immune cells and various subpopulations, cellular components including receptors, cytokines, immunoglubulins, and major histocompatibility (MHC) components have been reported. This complex network may be stimulated, which may lead to immunity or an autoimmune process, or suppressed by a myriad of chemical and biological agents.

The mucosal surfaces are in direct contact with the external environment and a major site of antigenic and toxic exposure in smokers. It has been reported that tobacco smoke contains over 4,000 toxic chemicals, many carcinogens ([15], [16]), as well as the potent immuno-modulator, lipopolysaccharide (LPS) ([17]). Toxins including endotoxin associated with tobacco smoke may impact inflammation contributing to coronary heart disease, as well as altering humoral and cell mediated immunity ([18], [19]). Understanding these effects prior to development of disease can be useful in understanding pathogenesis of the various pulmonary disorders and, perhaps, the overall systemic immune changes that occur in smokers.

This review focuses on and summarizes the results of studies of the immunologic response to tobacco smoking in healthy humans. The work done thus far will be organized based on the major systems within the immune response, namely humoral and cellular immunity.

HUMORAL IMMUNITY

Mucosal response

Secretory IgA (SIgA) is the main immunoglobulin isotype mediating humoral immunity in human secretions at luminal sites including oral, gastrointestinal, respiratory passages as well as in the eye. SIgA production is triggered after translocation of antigen from the lumen to mucosa-associated lymphoid tissue ([20]). The presence of IgG antibodies at the mucosal surface may be protective and it has been show that IgG2 subtype predominates ([21]). IgG in secretions is derived from serum or mucosa ([22]). Studies show tobacco smoke impacts both the systemic and mucosal immunity with changes demonstrated in mucosal antibody production and systemic antibody production. This includes passive smoking, which increases the risk of respiratory disease ([23], [24]).

A summary of studies evaluating mucosal immune response is shown in table 1. These studies generally suggest smokers have a decrease in salivary IgA (sIgA) levels, a form of SIgA ([25], [26], [27], [28], [29], [30]). This is especially seen in heavy (> 20 cigarettes/day) smokers ([14]) and may represent an acute inflammatory effect which resolves once smoking is stopped ([18]). Barton et al. found slightly higher salivary IgM in smokers, however, this is the only study to look for this finding ([28]). Nicotine was not a cause of elevated sIgA in this study.

Griesel et al. determined sIgA levels in people who stopped smoking for at least 2 weeks ([31]). Transient decrease in sIgA occurred followed by return to normal values within 2 weeks of stopping smoking. The level of sIgA in their study ranged from 3.5 mg/dL to 19.2 mg/dL. Evans et al controlled for social class, sex and age, and confirmed smoking to be an independent predictor of lower sIgA ([32]).

Serum antibody response

All five major classes of serum antibodies have been investigated in healthy smokers. The study results were variable as shown in Table 1.

Table 1 Smoking effects on humoral immunity

Author	Result	Comment
Secretory Response
Bennet (25)	1. sIgA—no difference	1. Seen in moderate smokers (20 cigs/20 yrs)
	2. Low sIgA	2. Highly defined smokers (20 cigs/40yrs)
Hersey (26)	Decreased sIgA
Jedrychowski (27)	Decreased sIgA and serum IgA
Barton (28)	Decreased sIgA Increased IgM in smokers
Ussher (29)	Decreased sIgA	Acute response (1 day), which returned to normal by 1 week after stopping smoking.
Olson (30)	sIgA—no difference	Evaluated effect of nicotine gum.
Griesel (31)	Decreased sIgA	Seen in first 2 weeks then returned to normal after stopping smoking.
Evans (32)	Decreased sIgA	Smoking is an independent predictor
Serum Immunoglobulin
Gerrard (42)	Decreased IgG Increased IgE	No difference for IgA, IgD, and IgM in smokers compared to non-smokers
Graswinckel (73)	No difference	Smoking significantly related to lower IgG and IgG2 in periodontitis
Gyllen (45)	Decreased IgG and IgG2	No effect of smokeless tobacco and nicotine
Qvarfordt (74)	Decreased IgG and IgG2	Seen in asymptomatic and smokers with chronic bronchitis. Chronic bronchitis patients had lower IgG4 than non-smokers.
Moszczynski (43, 44)	Decreased IgG, IgA, IgM	Long term (> 10 years) smoking effect
Wolfe (52)	Decreased IgA and IgG	No change in IgM

Many studies evaluated IgE levels in smokers ([33], [34], [35], [36]). Increased total IgE was found in both smokers and passive smokers ([37]). IgE elevation in smokers is particularly intriguing since IgE levels tend to decrease with age. Additionally, the increase in serum IgE in smokers is independent of allergic reactivity ([38]). Miyake et al. confirmed elevation of total IgE, but did not find elevation upon exposure to environmental tobacco smoke ([39]). Total IgE elevation was also reported in smokers by Court et al., and the authors noted an epidemiological pattern to elevated IgE levels that suggest a range for “usual” values ([40]). While total IgE elevation was confirmed for smokers, specific IgE (to house dust mite) was not increased. It has also been reported that smoking modifies genetic enhancement of total serum IgE levels ([41]). This is an intriguing report, but there are no other reports to confirm this finding.

Gender-related IgE response has been reported in smokers. A large study by Court 2002 evaluating more than 24,000 subjects found men have significantly higher IgE in both specific and non-specific IgE ([40]). Sherril et al. did not find any sex difference to explain smoking effects on IgE levels ([35]).

In 1980, Gerrard et al. showed lower serum levels of IgG and higher serum IgE in smokers compared to nonsmokers ([42]). The elevated IgE finding was not confirmed in a subset of teachers. The levels of IgA, IgD and IgM were not different. However, another study by Moszczynski showed decrease IgA, IgM and IgG without a change in absolute number or percent of B-cells ([43], [44]).

The mechanism of change in serum IgG level is not known, but there may be a dose-response relationshipwith lower levels of IgG occurring in those with increased smoking ([45]). Furthermore, nicotine was not observed to lead to a change in IgG level. Smoking may enhance underlying disease process in generation of disease specific immunoglobulin as seen in a study in asthmatics ([46]).

Systemic antibody response and development of influenza after vaccination was studied. Cruijff et al. examined the relationship between cigarette smoking and the occurrence of influenza, efficacy of influenza vaccination and antibody response after 3 weeks and 5 months ([47]). There was no relationship between smoking and either serological or clinical influenza. The risk of serological influenza was slightly, but not significantly, elevated in smokers compared to nonsmokers. The decline in serological titers after 5 months was similar for both smokers and nonsmokers. Thus, the authors conclude that smoking has no clinical or preventive risk of influenza in elderly. Smokers had an increased reactivity to influenza vaccine suggesting greater efficacy.

Finally, there were reports of elevated antigen specific immunoglobulins in smokers. This includes the elevation of specific immunoglobulin to a proteinase inhibitor ([48]), and elevation of Chlamydia pneumoniae specific IgA elevation in smokers ([49]). The elevation of the Ig specific anti-α 1 proteinase inhibitor was seen in patients with rheumatoid arthritis, was independent of the underlying disease and related to smoking status. The significance of these findings is not known.

In summary, studies results for secretory, specifically salivary IgA, are mixed. The majority of the studies show decreased in sIgA levels and one study demonstrating the decrease in sIgA as an acute response. The decrease in sIgA may be dose dependent as seen in Bennet et al., where the decrease occurred in heavy smokers (> 20 cigarettes/day). Clinical significance of sIgA change is not known, that is, whether this translates to increased risk of local or systemic infection. Overall, the studies evaluating serum immunoglobulins show a decrease in serum IgA and IgG with one study reporting decreased serum IgM. Serum IgG2 decrease has also been reported and may explain increased respiratory tract infections when considered with the decreased IgA ([45], [74]). Additionally, serum IgE level is increase in smokers. Last, no alteration in specific antibody immunity has been consistently shown.

CELLULAR IMMUNITY

The large number of toxins in cigarette smoke places smokers at risk for immune suppression and diseases including cancer and viral infections. This raises the question as to what are the changes in cell mediate immunity related to smoking. Are the smoke constituents stimulatory or inhibitory? Which cell lines are affected, what are the effects on regulatory cells and cytokines?

In 1998 Morrison found increased number of cells in the broncho-alveolar lavage fluid (BALF) from acute smoking ([50]). The total number of cells recovered was 6.5 times greater in acute smokers compared to nonsmokers. The percentage of neutrophils in BALF from the acute smoking group compared to the nonsmoking group was also higher. Both macrophage and lymphocytes results did not differ between acute smokers and the nonsmoking group. Thus, acute inflammatory response occurs as a result of acute injury and is likely sustained by repeated insults to the lung with continued smoking. Yamaguchi et al. found decreased lymphocytes in the BALF of smokers compared to that of non-smokers, but this was not statistically significant (14 vs 10 × 10³/ml fluid) ([51]). Overall, the current literature search provides very little information on cell counts from BALF in smokers without clinical pulmonary disease.

There was one study providing information on peripheral blood lymphocytes in smokers and non-smokers. The study assessed various immunological parameters in 497 men in the U.S. Air Force ([52]). Twenty-seven percent never smoked and 33% of the men were smoking at the time of the study. This study did not present data on the presence or absence of pulmonary disease in the smokers. Overall, active smoking increased all immune cell numbers including total lymphocytes, total T-cells, helper and suppressor T-cells, B-cells, and monocytes. The author did suggest age-specific ranges for these cell populations should be determined.

Effects on cytokines

Cytokines are a diverse family of soluble modulatory proteins and peptides which act as regulatory agents ([53]). They are secreted by various cells in the body including white blood cells, macrophages, fibroblasts and epithelial cells. Cytokines enable and provide communication between cells and have various effects on cellular proliferation, growth, differentiation, homeostasis and other functions. Table 2 list known cytokines, provides a summary of the source of each cytokine and general function of each class, and a summary of specific cytokines in selected cases.

Table 2 Cytokine families and members ([53])

Family	Members	Principal Source	General Effect(s)
IL-1	IL-1α /IL-1F1 IL1-β /IL1-F2 IL-1 receptor-antagonist/IL-1F3 IL-18/IL-1F4	Macrophages, antigen presenting cells Dendritic cells mononuclear/macrophages	Primarily proinflammatory cytokines; initiation of COX-2, type 2 phospholipase A and inducible nitric oxide synthase (iNOS). Increase expression of adhesion molecules; angiogenic factor; aids differentiation of marrow stem cells in myeloid series. IL-18 induces INFγ, IL-4, IL-5 and IL-13 in concert with other factors.
IL-2/IL-4	IL-2 IL-3 IL-4 IL-5 GM-CSF	TH-1 cells, NK cells T-cells, macrophages TH-2 cells, mast cell TH-2 cells, mast cells T-cells, macrophages, fibroblasts	Pleiotrophic actions including augment proliferation of T-cells, role in activation induced cell death, promote growth and differentiation B-cells, augment NK-cell cytolytic activity, induces lymphokine activated killer activity and proliferation of large granular lymphocytes; induces IL-2 receptor; and stimulate monocyte cytotoxicity, GM-CSF, IL-1β and IL-6 mRNA and protein. IL-3 has very broad cell targets and stimulates generation and differentiation of dendritic, macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes and erythroid cells. IL-4 induces biological functions on dendritic cells and T-cells. IL-5 eosinophil hematopoietic growth factor, B-cell growth and differentiation factor GM-CSF stimulates growth of multiple myeloid cells, augments function of phagocytes, macrophages, dendritic cells and eosinophils
IL-6/IL-12	IL-6 IL-12 IL-23 IL-27	TH-2, antigen processing cells, somatic cells B-cells, macrophages APC, dendritic cells, macrophages, T-cells, monocytes TH-1, monocytes, macrophages Monocytes, DC, placenta	IL-6 pleiotropic cytokine that regulates immune reactivity, acute-phase response, inflammation, oncogenesis and hematopoiesis. IL-12 pleiotropic immuno-modulation TH-1 response inducer, enhance B-cell survival but suppresses IgE synthesis. IL-23 IL-12 like-effects, activate memory T-cells, increase acute phase proteins, IL-1, TNFα, INFγ. IL-27 function not fully clear; role inducing TH1 immunity
	IL-7	Thymic, bone marrow and hepatic stroma, smooth muscle, fibroblast, dendritic cells, endothelial cells	Growth and differentiation factor for γ δ T-cells, B-cell development, promotes effector functions of T-cells, NK cells, monocytes.
	IL-9 IL-11, IL-13, IL-14 IL-16	T-cells Fibroblasts, epithelial cells, chondrocytes, synoviocytes, endothelial cells, osteoblasts T-cells, NK, basophils, eosinophils, mast cells, dendritic cells T-cells, mast cells eosinophils, DC, B-cells, bronchial epithelial, fibroblast	IL-9 T-cell and mast cell growth factor; synergize B-cell IgE and IgG production; modest hematopoietic activity. IL-11 hematopoietic (thrombopoiesis) cytokine, protects and restores gastrointestinal mucosa; immuno-modulating agent, affects bone metabolism. IL-13 immuno-modulation B-cells with role in effector phase of allergic and asthma response protects from helminthes infections. IL-14 is high molecular weigh B-cell growth factor. IL-16 T cell chemoattractant, proliferation and differentiation.
IL-10	IL-10 IL-19 IL-20 IL-22 IL-24 IL-26 IL-28 (IFNλ 2, 3) IL-29 (IFNλ 1)	TH-2 cells, activated T-cells and B-cells, monocytes/macrophages, mast cells, keratinocytes	Conti et al([75]) Regulator of immune response; turns off T-cell cytokine production; inhibits monocyte/macrophage production of IL-α 1, LI-1β, IL-6, IL-8, IL-12, TNF, GM-CSF, INF and other cellular functions. IL-19 induce IL-10([76]). IL-20 promotes keratinocyte proliferation. IL-22 promotes acute phase proteins production, inhibit IL-4 production. IL-24 suppresses tumor growth; linked to wound healing. IL-26 function not defined.
IL-15	IL-15 IL-21	Macrophages, antigen presenting cells CD4 T-cells	IL-15 shares activity with IL-2, stimulates T-cells, B-cells, NK cells, monocytes, eosinophils, neutrophils, mast cells, non-immune cells; pivotal role in immune response, graft rejection and autoimmunity IL-21 immunoregulatory function for B-cells, T-cells; enhance NK cell effector function
IL-17	IL-17 IL-25	memory T-cells	Mediate the stimulation of neutrophil mobilization, induces many other cells to produce various cytokines. IL-25 increase cytokines, IgG1 IgE and IgE, eosinophilia.
Newer interleukins	IL-30 IL-31 IL-32	T-cells Mononuclear, NK cells	P-28 subunit of IL-27 Induce chemokines Induces TNFα, IL-8 and MIP-2CXCL-2
Type I Interferons (IFN) Type II IFN	IFN-α IFN-β IFN-ε IFN-κ IFN-ω INF-τ IFNλ IFNδ IFN-γ	Macrophage and neutrophils TH-1, NK cells, fibroblasts keratinocytes leukocyte Trophoblast Dendritic cells T-cells, NK cells	Anti-viral, pro-apoptotic, proliferation and maturation of NK cells, induce IL-15 and IL-10, inhibit T-cell migration, and promote T-cell survival. Promotes innate and adaptive immunity to infectious agents (anti-viral) and tumors; promotes antigen processing and presentation; regulates B-cell functions; role in TH1 development.
Tumor necrosis factors	TNF-α TNF-β /LT-α LT-β Fas ligand CD-40 ligand TRAIL BAFF APRIL RANK LIGHT	Various immune and non-immune cells	Pleiotropic pro-inflammatory cytokine with multiple biological effects at cellular and organ level.
TGF-β	TGF-β Bone morphogenetic proteins Inhibins Activins	Immune and non-immune cells	Inhibitor of cell proliferation, immunosuppressor, pro-inflammatory, anti-inflammatory, tissue remodeling
Chemokines	CXC subfamily (CXCL1-16) CC subfamily (CCL1-28) C subfamily (CL1 / Lymphotactin) CX3C subfamily (CX3CL1 / Fractalkin) IL-8 (CXCL8)	All somatic cells Peripheral blood mononuclear, T-cells, neutrophil, endothelial, fibroblast, epithelial, NK cells	Pro-inflammatory mediators with chemotactic activity for spectrum of cells

Interleukins are cytokines originally thought to be produced by white blood cells and key effector proteins in intercellular communication between cells of hematopoietic origin. As the specific identification and source of production became clear, the name “interleukin” did not adhere to original definitions. “Interleukins” will be used based on specific regulatory factor name and “cytokine” will be used to refer to the overall family of cytokines. The number of interleukins continues to grow, and there are now over 30 interleukins.

The lung is directly exposed to the environment and must protect itself from a host of insults including microbial, allergens and toxins. Protection of the lung occurs through various mechanisms including structural components, as well as cough reflex, mucociliary clearance, and the presence of both immune and non-immune cells that elicit protection and recruit cells to eradicate and neutralize noxious agents. Importantly, cytokines, based on their role in cellular recruitment, proliferation, regulation of growth factors, play a key role in the innate and adaptive immune response in the lung ([54]).

The majority of the quantitative studies on cytokines in smokers were performed on BALF. Studies evaluating cytokine levels in sputum and peripheral blood are few in number (Table 3). The studies found for this review addressed cytokine production in COPD; however, control populations included “healthy smokers” and non-smokers. This review focuses on cytokines changes in healthy smokers.

Table 3 Summary of cytokine changes found in healthy smokers

Specimen	Cytokine	Smoking effect	Comments
BALF
	IL-1	Less IL-1	Yamaguchi ([51]) Stimulated alveolar macrophages
	IL-2 IL-4 IFN-γ	Decreased Indeterminate Decreased	Hagiwara ([77]) Mitogen stimulate cells; heavy smokers
	IL-1β IL-6 IL-8 MCP	Increased Increased Increased Increased	Kuschner ([58]) Study demonstrated dose response effect of smoking in cells counts as well as cytokines
	IL-1ra, IL-6, IL-8 TNF and IL-1β	Decreased Decreased Decreased No difference	Mikuniya ([59]) BALF stimulated macrophages. Il-1ra and IL-6 most prominent changes
	IL-6 IL-8 TNF	Decreased No difference Decreased	McCrea ([57]) LPS stimulated macrophages
	IL-1β IL-6 IL-8 TNF-α IFN-γ	Decreased Decreased No difference Decrease Decrease	Alveolar macrophages
	IL-1 IL-6	Decreased Less	Soliman ([63]) LPS induced Intracellular IL-1 high; undetected IL-6 in cell in both
	IL-8	Increased in BALF, Decreased	Ohta ([60]) Stimulated alveolar macrophages
	IL-8	No difference/ increase in acute smokers	Morrison ([62]) Neutrophil source
	IL-16	Increased	Laan ([65]); Smokers > Non-smokers; no difference between types of smokers. Negative correlation with CD 4 number of cells
Sputum
	IL-10	Decreased	Takanashi ([68])
	TNF-α sTNF-R IL-8	No difference Increased Increased	Vernooy ([66]) Sputa had more inflammatory cells and less macrophages in diseased vs. healthy smokers
	IL-18	Decreased	McKay ([69])
Peripheral blood
	IL-4 IFN-γ	No difference between smokers and non-smokers	Majori ([70])
	IL-4 IFN-γ	Increased No difference	Byron ([71]) phytohemagglutinin stimulated PBMC
	IL-6 TNF-α	Less	Wirtz ([72]) LPS stimulated WBC
	IL-10	Less Decreased	Karjalinen ([56]) IL-10 haplotype assoc with smoking and lung impairment, and regulation of IgE production. They posed the gene may differentially respond to disease vs. normal/control situation.

Bronchoalveolar studies

In 1989 Yamaguchi reported decreased IL-1 production by alveolar macrophages (AM) from smokers ([51]). The AM were obtained by brochoalveolar lavage, and then cultured. Both the AM cell culture supernatant and supernatant from LPS stimulated AM were evaluated by a bioassay for IL-1 activity. This is one of the earliest reports demonstrating a cytokine production defect in the pulmonary environment of smokers (decreased proportion of lymphocytes in BALF and reduced CD4/8 ratio was also reported). The decrease IL-1 was due to decreased “capacity” of AM cells and not due to inhibition by prostaglandins which was confirmed by using indomethacin. Furthermore, they confirmed results by using enzyme-linked immunosorbent assay for IL-1β.

IL-1 DNA haplotype determined from peripheral blood may be associated with a decline in lung function in smokers ([55]). Joos et al. reported a decline in lung function in smokers associate with IL-1 gene haplotypes. A study by Karjalainen reported IL-1 haplotypes were significantly associated with the rate of decline of lung function in asthmatics, but not controls ([56]). Thus, work by Karjalainen utilizing the same gene polymorphism and same methods in adult non-smoking asthmatics and non-smoking controls indicated the finding in the study by Joos is not limited to smokers.

Additionally, the rate of decline in lung function was the opposite in smoker as compared to asthmatic. The haplotype associated with rapid decline in pulmonary function in the Joos cohort was associated with slow decline in asthma in Karjalainen study; and the haplotype associated with slow decline in pulmonary function in Joos' study was associated with rapid decline in asthma in Karjalainen study. This does not negate the findings, but raises the interesting prospect of variation of cytokine and/or genetic response in different disease process which is modulated or expressed based on other as yet undetermined factors.

As seen in Table 3, the majority of studies on BALF and sputa focused on chemokines including IL-8, MCP-1 (CCL-2) and IL-18. This research was likely driven by:

1. Knowledge of the role in recruitment and activation of neutrophils and monocytes in the airway by IL-8;

2. Induction of IL-8 by TNF-α;

3. Possible role of IL-6 in inhibiting inflammation.

McCrea et al. showed BALF in healthy smokers had lower IL-6, but no difference in IL-8 compared to nonsmokers. Furthermore, LPS stimulated macrophages released less TNF and IL-6 ([57]). This was in the presence of smokers having significantly higher number of cells. Two smokers with the highest BALF cell counts had elevated IL-8. Kuschner also found increased number of cells, including macrophages ([58]). The proinflammatory cytokine IL-8 was significantly elevated and its release demonstrated a dose response to cigarette smoking (up to 2 ppd). Secondly, epithelial cells may play role in cytokine release. Mikuniya found no difference in smokers compared to nonsmokers in levels of supernatant IL1β, TNF-α, and IL-8. However, stimulated macrophages had decreased IL-1ra and IL-6([59]). The decrease IL-1ra also occurred in unstimulated macrophages. Thus, the results appear to be mixed, but stimulatory assays for IL-1, IL-6 and TNF suggest decreased capacity to produce cytokines. Further studies are needed on both IL-1 and IL-8.

Ohta et al. also showed an increased in BALF and decrease in LPS stimulated macrophages IL-8 secretion. Peripheral blood mononuclear cells, stimulated and unstimulated, produced comparable levels of IL-8 ([60]).

Kotani et al. evaluated BALF obtained from orthopedic surgical patients under anesthesia. This study provides useful information regarding gene expression in AM for IL-1β, IL-6, IL-8, IFN-γ, and TNF-α ([61]). This study demonstrated decrease in pro-inflammatory cytokines and showed no difference for the regulatory cytokine IL-8. IL-6 production may require the presence of epithelial cells as well as IL-8 production; however, one cannot ignore the potential role of anesthesia. Additionally, decrease mRNA for all pro-inflammatory cytokines was seen in smokers, with the exception of no difference in IL-6 between groups or change from baseline.

Morrison et al. ([62]) found the same results as McCrea for IL-8. There was no difference in the presence of IL-8, but an increase was seen in LPS stimulated BALF cells in acute smoking (within 1 hour of broncho-alveolar lavage), which further supports the idea of dose effect. A similar finding for the neutrophil chemo-attractants, growth-related oncogene-α and extractable nuclear antigen, was seen in the same study.

Soliman et al. added more issues ([63]). A decreased level of IL-1 and IL-6 was seen in LPS stimulated AM. There was increased intracellular IL-1 however, and the study demonstrated a lack of secretion of IL-1 as previously reported by Brown et al. ([64]). They further demonstrated that some smokers had an inhibitor to IL-6. Thus, in some smokers the antigenic IL-6 level was not different from nonsmokers, but bioactive IL-6 was clearly decreased. Hence, a mixed picture occurs that is possibly explained by changes in the regulatory pathway with production of an inhibitor to IL-6.

Last, Laan and colleagues evaluated IL-16 levels in BALF and peripheral blood lymphocytes ([65]). It is a selective CD4+ T-cell chemoattractant and induces IL-2 receptor expression by airway epithelium and CD8+ T-cells. IL-16 was increased in smokers. This later finding is opposed to population based findings in smokers regarding decreased CD4 numbers and function peripherally.

Sputum studies

Sputa studies on the presence and cytokines in general have been few. Vernooy found increased IL-8 which was partially explained by increased presence of neutrophils ([66]). The TNF-α level was not different between smokers and non-smokers. Soluble TNF receptor levels in sputa and peripheral blood were also investigated. TNF-receptors of epithelial origin were less and there was no difference in the TNF receptor of myeloid origin. The study report also presented results of peripheral blood TNF-receptor of epithelial origin, which did not show any difference between healthy smokers and smokers with COPD.

However, the soluble TNF-receptor level was significantly higher in plasma of COPD patients. It is difficult to draw a conclusion since no data on normal controls were presented. The data on plasma level showed no IL-8 or TNF-α peripherally in healthy smoker. This study was small containing 17 healthy smokers compared to 18 patients with COPD and variable smoking status. This study does highlight a differential effect on cytokine and receptor level in the local (lung) inflammatory site as compared to the systemic findings.

Schulz et al. evaluation of IL-8, TNF-alpha, and IFN-gamma did not show any difference between healthy smokers and controls from BALF, sputa, or primary bronchial epithelial cells ([67]). IL-10 is an important regulatory cytokine that reduces inflammatory response/cytokine production by eosinophils. IL-10 was lower in smokers compared to nonsmokers. Further work showed this was not due to clearance and suggests decreased cytokine production ([68]).

IL-18, originally called an IFN-γ inducing factor, is pro-inflammatory and secreted by airway epithelia and macrophages. While the McKay study was performed to evaluate asthmatics, data are presented for healthy smokers and nonsmokers ([69]). IL-18 level was decreased in healthy smokers as compared to non-smokers. IL-18 mRNA expression in cells from sputum did not significantly differ between smokers and non-smokers and the level of IL-18 in healthy smokers may resemble that seen in stable asthmatics. This result is very intriguing and opens the door to many possibilities for the alteration in production of IL-18 ranging from defects in production to inhibition of production of IL-18. Perhaps there is some altered form of the cytokine leading to increased destruction or elimination.

Peripheral blood studies

There were a few studies evaluating cytokine changes in peripheral blood. A total of 4 studies were found in the literature. Majori et al. evaluated TH-1/TH-2 intracellular cytokine production in subjects with COPD, and also provided results for healthy smokers ([70]). No difference in percentage or absolute numbers of CD3, CD19, CD23 cells, as well as CD4, CD8 cell populations was seen between the healthy smokers and those with overt disease. Cytokine production had to be stimulated in order to detect their presence. INF-γ and IL-4 were not spontaneously present. While the %CD4+ cells with IFN or IL-4 showed a difference between healthy smokers and those with COPD, a normal control population was not available to compare the results of the healthy smokers.

The findings by Miller et al. ([78]) on peripheral blood immunoregulatory T-cells in smokers differed from the findings of Majori. Total T-cell numbers were not different across non-smokers, light (10–19 pack-years), moderate (20–49 pack-years) and heavy smokers (50–120 pack-years). There were less CD4, but increased CD8 lymphocytes. The Miller study was an early report and functional data for the lymphocytes are not available. However, this study suggests, as seen previously in other studies, the possibility of dose-effect phenomena of smoking. The subjects in this study were evaluated for serious illness, immunodeficiency and use of immunosuppressive drugs, however, no data were provided regarding the pulmonary status. Thus, it is not clear that there was no significant pulmonary obstructive disease. The reverse CD4/CD8 ratio was not seen in peripheral blood lymphocytes, but present in BAL cell population in a small study (n = 12 smokers) by Costabel ([79]).

Byron et al. studied IL-4 and IFN-γ production in peripheral blood mononuclear cells (PBMC) using phytohemagglutinin stimulation ([71]). Again, no difference was seen in the production of IFN-γ, but more IL-4 production was found in smokers compared to non-smokers, particularly the heavy smokers. Wirtz and colleagues showed decrease IL-6 and a trend for higher TNF among smokers whose leukocytes were stimulated with LPS ([72]). However, this study only included men.

Immunopathology findings

Finally, there was one immunopathology study of airway subepithelium in smokers with and without COPD ([80]). As seen in studies using peripheral blood and BAL, there were increased numbers of CD8 cells. This finding was only seen when comparing the smokers without COPD to those with COPD. There was no difference in CD8 cell counts when non-smokers were compared to either smoking group. There was no significant difference in tissue eosinophils, CD4, total T-cells or activated T-cells. Interestingly, there was a negative correlation between activated eosinophils as measure by presence of major basic protein and FEV₁, and a positive correlation in pack-years and the presence of CD8 cells.

SUMMARY/CONCLUSION

The study results in both healthy and diseased smokers represent a beginning to building a pathogenetic picture of immune reactivity in tobacco smokers. First, the lung as a major and significant immune organ is demonstrated by studies summarized in this review. Second, with the introduction of a considerable number of toxins with local tissue damage and activation of local immune cells including macrophages, epithelial cells, fibroblast, and dendritic cells, both non-specific and specific immune mechanisms are initiated. A host of chemokines and interleukins are produced locally to mediate this activation and recruitment process including the production of inhibitory regulatory factors. Granulocytes and lymphocytes may recruit through mediators as well as through the activation of circulating immune cells within the local vascular and perivascular areas. The circulating activated inflammatory and immune cells can further amplify the immune response in the peripheral blood stream. Although not summarize here, non-specific proteases and reactive oxygen species are produced leading to neutralization of toxins and damage to local tissues as the inflammatory process persist or escalates.

The work on humoral immunologic changes appears clear pointing to a decrease in sIgA and, therefore, decreased airway protection from infection. The immune surveillance defect may be further heightened by the decreased in serum immunoglobulins of smokers, but the significance of peripheral blood immunoglobulin changes is not clear. Further research is needed. The association of quantitative antibody changes in smokers with infection is not clear as shown in Qvarfordt paper on chronic bronchitis. Tobacco smoking has the ability to enhance or suppress antibody response dependent upon the number of cigarettes and perhaps duration of smoking regardless of the number of cigarettes per day. Study populations need clarification particularly with regard to the extent of lung disease. The increase in non-specific IgE is interesting and may represent immunoglobulin class switch stimulated by LPS in tobacco smoke ([81]). The increased IgE could play a role in mast cell mediator release in the overall inflammatory response.

Changes in cytokines represent areas of interest and importance. The changes in cytokine production in healthy smokers seem clear for IL-6, IL-1 and TNF, which generally decrease. These results suggest less inflammation and therefore less tissue damage in healthy smokers. IL-8 results are mixed. It would be useful and important to confirm these results as IL-6, IL-8, IL-1α, and IL-1β are key inflammatory mediators associated with tissue damage. Schulz et al. ([67]) has shown increased constitutive and simulated release of IL-8 in patients with chronic obstructive pulmonary disease, but are there earlier points within the activation sequence of turning on the expression and/or secretion of IL-8 in healthy smokers? Are other inhibitory signal or proteins present as seen with IL-6 and what are the stimuli for these inhibitors? IL-13, a potent stimulator of B-cells especially in allergic individuals, may be present in smokers as an explanation for some of the immunoglobulin findings noted above.

Overall, a key question exists as to the inter-relationship of local pulmonary cytokines, whether found in sputa, BALF, or stimulated mononuclear cells obtained from the lung to that of peripheral blood cytokines remains. What are the sources of cytokines, how are they processed, what are the kinetics? What is/are the primary inflammatory stimulus (stimuli) in smoke? Many questions remain in this area and this may be of importance in understanding disease pathogenesis for infection, malignancy or atherosclerosis. For any toxin the primary treatment is avoidance. Large strides are being made in the United States on smoking education and abstinence, but significant numbers of smokers, many young smokers, remain.