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Department of Medicine, Division of Pulmonary and Critical Care, Indiana University Medical Center, Indianapolis, Indiana
Correspondence and requests for reprints should be addressed to Homer L. Twigg III, M.D., Associate Professor of Medicine, Indiana University Medical Center, Richard Roudebush VA Medical Center, 1481 West 10th Street, VA 111P-IU, Indianapolis, IN 46202-2884. E-mail: htwig{at}iupui.edu
ABSTRACT
The humoral, or antibody, immune response is essential for host defense against bacterial pathogens. The lung has the ability to respond quickly to some pathogens through stimulation of resident antigen-specific memory B cells. Alternatively, after exposure to a new pathogen, the lung can generate de novo both a systemic and local (mucosal) antibody response. The resulting production of antigen-specific IgG and IgA act in concert to help clear the invading pathogen and reduce subsequent colonization of respiratory epithelium. Systemic vaccination against respiratory pathogens, although effective in generating systemic IgG responses and some mucosal IgA responses, may be less effective than vaccination through mucosal surfaces, which induce a brisk IgA and IgG response both locally and systemically depending on the site of antigen deposition. The local response is important in reducing colonization of the upper respiratory tract by pathogenic bacteria, the first step in the development of most causes of pneumonia. Future studies are needed to provide further insight on the site of pulmonary humoral host responses to bacterial challenge and optimal vaccine regimens to minimize the burden of respiratory disease caused by pathogenic bacteria.
Key Words: antibody • bacterial pneumonia • immunoglobulin • vaccine
Humoral, or antibody-mediated, immunity is essential for host defense against bacterial pathogens. Patients with defects in humoral immunity are primarily susceptible to recurrent bacterial sinopulmonary infections and bronchiectasis (1–3). This article focuses on recent advances in the understanding of the normal B-cell environment in the lung, generation of appropriate antibody responses after antigenic challenge, and the pulmonary response to vaccination. Respiratory infections and vaccines, especially pneumococcal, are emphasized to illustrate the lung response after exposure to bacterial antigens. Some of the data shown to support concepts described in this article have been previously reported in the form of an abstract (4).
NORMAL B CELL AND ANTIBODY CONCENTRATIONS IN THE LUNG
The primary cell responsible for generating humoral immunity is the B lymphocyte. B lymphocytes comprise 1 to 10% of the lung lymphocyte population and can be separated into two main classes. Plasma cells constitutively secrete IgG and other immunoglobulin subclasses (5, 6). In contrast, memory B cells produce immunoglobulin only in response to reexposure to particular antigens (6). B cells can be further classified into B1 and B2 cells. B1 cells were first described in the gastrointestinal tract. These cells are IgM+, CD5+ cells that do not require T cell help for development (7). In the lamina propria B1 cells undergo class switching to an IgA secretory cell. B1 cells are thought to be important in innate immunity against conserved bacterial antigens. Because of their autonomous ability to secrete antibody, they are also believed to contribute to autoimmune diseases. These cells are difficult to demonstrate in normal lung. In contrast to B1 cells, B2 cells require T cell help, mainly through secretion of IL-4, IL-5, IL-6, and IL-10, and ligation between CD40 on B cells and CD40L on T cells (8).
The relative proportion of immunoglobulin present in the lower respiratory tract differs dramatically from that of the upper respiratory tract. Immunoglobulins comprise the second largest class of proteins present in bronchoalveolar lavage (BAL) fluid after albumin (9). IgG is of primary importance in the lower respiratory tract (10). Smaller amounts of IgA and IgE are consistently found in the BAL fluid of normals (9). The opsonizing and complement activating properties of IgG dramatically aid phagocytic cells in clearing invading microorganisms. The four IgG subclasses exist in BAL fluid in approximately the same proportions as found in serum. IgG1 represents approximately 60 to 70% of the IgG present in BAL fluid, IgG2 20 to 25%, and IgG3 and IgG4 are present only in small amounts (< 5%) (10). Responses to protein antigens predominantly reside in the IgG1 subclass, whereas polysaccharide antigens predominantly give rise to IgG2 antibodies (3, 11).
In contrast to the lower respiratory tract, the upper respiratory tract contains predominantly IgA with lesser amounts of IgG. IgA is the most abundant immunoglobulin in secretions and in the upper respiratory tract exceeds the concentration of IgG by a ratio of 2.5:1. IgA has two subclasses: IgA1 and IgA2 (12). IgA1 comprises nearly 80% of serum IgA. In contrast, IgA2 seems to be important in mucosal immunity and nearly half of the IgA present in secretions is IgA2. IgA exerts its protective effect through three mechanisms (13). First, it serves as an immunologic barrier, inhibiting binding of organisms to mucosal surfaces. Second, the normal movement of IgA from the basilar to apical region of epithelial cells suggests that it may be effective in neutralizing intracellular pathogens. Finally, pathogens bound to IgA may be taken up by airway macrophages through the phagocytic process.
Most B cells in the lung are mature memory B cells. This mirrors the predominance of memory T cells in the lung (14), suggesting that the lung is primed to respond to antigenic challenges at a moments notice. IgG-, IgM-, and IgA-secreting B cells are present in normal subjects; however, poor correlations exist between the numbers of immunoglobulin producing BAL cells and the levels of immunoglobulin in BAL fluids. In BAL fluid from nonsmokers IgG is present in the same proportion as in serum, leading to speculation that most IgG enters by transudation from the plasma compartments (9, 15). These two observations fit in well with the primary presence of memory B cells in the lung, suggesting that under resting conditions very little local antibody is made. Only after antigenic challenge is local production increased. Our lab and others have found increased ratios of specific to total antibody in BAL fluid after pneumococcal infection suggesting that local antibody production can be increased after relevant exposures (16). In contrast to IgG, the concentration of IgA adjusted for total protein in lung fluid is greater than that of serum under normal conditions, suggesting that IgA is locally produced (17).
GENERATION OF AN ANTIBODY RESPONSE AFTER ANTIGENIC EXPOSURE
Much of what is known about pulmonary humoral immune responses comes from work of Kaltreider and Curtis in the late 1980s (Figure 1). Using intratracheally administered sheep red blood cells in a mouse model (18–20), these investigators proposed a model wherein particulate antigen is taken up in alveolar space by alveolar macrophages or other antigen-presenting cells. These cells then migrate by lymphatics to regional lymph nodes where the primary immune response occurs. Once antigen-specific B cells are formed they traffic back to lung where they terminally differentiate and expand into either antibody-secreting plasma cells or memory B cells. Importantly, in their model lymphocyte and antibody responses in BAL fluid are identical to that found in lung tissue (18), suggesting that BAL may be an accurate marker of pulmonary humoral responses.
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Once antibody-producing B cells are formed in secondary lymphoid tissue or mucosal-associated lymphoid tissue, they must traffic back to the original point of entry of the pathogen. This trafficking of lymphocytes back to mucosal sites has been intensely studied, giving rise to a four-step model. The first step is tethering. L-selectin on lymphocytes interacts with addressins on endothelial cells to slow down the movement of lymphocytes through capillaries. In nasal-associated lymphoid tissue, the responsible endothelial receptor is PNAd (25), whereas in the lung adhesion is mediated by intercellular adhesion molecule-1 (26). Tethering leads to lymphocyte activation mediated by chemokines and their receptors and subsequent firm adhesion to the endothelium. The latter is mediated by leukocyte function–associated antigen-1 binding to intercellular adhesion molecule-1 and 
4ß7 integrin binding to MADCAM-1 (27). Finally, diapedesis through the mucosa occurs, a process that probably involves all the previously mentioned receptor–counter receptor interactions.
It is relatively easy to demonstrate increased pathogen- specific antibody in the serum after infection. If there is truly local antibody production in the lung after pulmonary infections, however, there should be increased local secretion of specific antibody in the lung after antigenic challenge. Our lab has demonstrated significantly increased pneumococcal antibody titers in the lungs of normal and HIV-infected subjects who have had invasive pneumococcal infection (16). In these subjects, the ratio of pneumococcal-specific to total IgG was greater in the lung than in plasma, suggesting localized production of antibody in the lung.
Finally, once antibody is produced in the lung, it must be effective in promoting clearance of the offending pathogen. Just having a vigorous antibody response is insufficient. For example, HIV infection is characterized by an increased amount of total and pneumococcal-specific antibody in the alveolar space (16, 28), yet invasive pneumococcal disease is prevalent in this population. We have recently found that HIV-infected Malawians, a population with a high incidence of invasive pneumococcal infection, have four times the amount of pneumococcal-specific IgG in BAL compared with non–HIV-infected subjects. The ability of highly purified BAL IgG from HIV-infected subjects to bind pneumococci is impaired, however, compared with BAL IgG from non–HIV-infected subjects (Figure 2) (4). This population seems to have dysfunctional pneumococcal antibody in the alveolar space. The reasons for this observation are not known, but could include structural abnormalities in HIV IgG or the lack of a diverse antigenic response (i.e., antibody produced against only a few pneumococcal antigens in HIV-infection compared with a more diverse response in non–HIV-infected subjects) (29). These findings highlight the recently recognized fact that better correlates are needed for assessing the host response to natural infection or vaccination besides simple measurement of antibody concentrations (30).
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Vaccination against pulmonary pathogens is a common and effective practice against many diseases. Mechanisms behind their effectiveness, however, are not always clear. In theory, both IgA and IgG have a role in protection after vaccination. The presence of pathogen-specific IgA should decrease colonization of the respiratory tract by limiting attachment to respiratory epithelium. Because airway colonization is usually the first step in the development of bacterial pneumonia, decreased colonization should lead to a decreased incidence of pneumonia. When pathogens manage to reach the alveolar space, IgG should take on a greater role as an opsonin in the phagocytosis of organisms. The most effective immunity against bacterial infections should involve both and IgG and IgA response. That being said, the vaccine delivery mechanism impacts the type of immune response generated. In general, systemic administration of a protein antigen results in the generation of circulating IgG, some of which diffuses into the epithelial lining fluid. In contrast, mucosal antigenic challenge results in a more vigorous IgA response locally at the site of challenge. This section examines what is known about the pulmonary response to vaccination.
Despite the recognition that local antibody may be of paramount importance in pulmonary host defense, there is remarkably little information on humoral responses in the respiratory tract after systemic immunization, the standard approach to vaccine strategies. Much of what is known about mucosal immunity comes from animal models. In a mouse model of mucosal immunity against Mycoplasma pulmonis, it has been demonstrated that the site of antigen deposition greatly influences the antibody response and subsequent protective immunity (31). Intranasal challenge resulted in an increase in IgA antibody–forming cells in the nasal submucosa and an increase in mycoplasma-specific IgA in nasal washes. Low numbers of IgA antibody–forming cells and IgA concentrations were seen in the lung after isolated nasal challenge, but were increased significantly if animals received both nasal and pulmonary immunization. Both nasal and nasal-pulmonary vaccine exposures resulted in equivalent antigen-specific IgA and IgG in the systemic circulation. Both forms of immunization also significantly reduced the ability of mycoplasma to colonize the nasal passages after experimental exposure to a mycoplasma inoculum. Animals receiving nasal-pulmonary immunization, however, had significantly fewer organisms in the lungs compared with animals that only received nasal immunization. Because serum mycoplasma-specific IgA and IgG were similar after both types of immunization, this suggests that circulating antibody is less effective in protection against pulmonary pathogens compared with locally produced antibody.
In another animal immunization model against a different pulmonary pathogen, Moraxella catarrhalis, Jiao and colleagues examined the site of specific antibody production and protection after intranasal and subcutaneous challenge (32). Intranasal administration of M. catarrhalis surface proteins resulted in antigen-specific IgA and IgG in both nasal washes and BAL. IgA concentrations far exceeded IgG concentrations in both sites. IgA was significantly higher in BAL and nasal washes than in serum, whereas IgG was higher in serum than both mucosal sites. Immunization was associated with a marked increase in bacterial clearance after intranasal challenge with live M. catarrhalis organisms. As might be predicted, subcutaneous administration of the same vaccine resulted in production of far greater antigen-specific IgG compared with IgA, especially in the lung, where Moraxella-specific IgG in BAL was nearly 20 times greater than that found in nasal washes. Subcutaneous vaccination induced a marked systemic IgG response, but virtually no antigen-specific IgA in serum, BAL, or nasal washes. Interestingly, despite the brisk systemic and pulmonary IgG response after subcutaneous immunization, intranasal immunization promoted significantly greater bacterial clearance after intranasal challenge with the organisms. These data further support the general premise that whereas systemic vaccination results in system antigen-specific IgG, which may passively diffuse into the epithelial lining fluid in the lung, significant protection is only afforded if local IgA immune responses are also induced.
The effect of the polysaccharide pneumococcal vaccine has also been studied in animal models. Lynch and colleagues have examined the effect of intramuscular and intranasal administration of the pneumococcal vaccine in a mouse model (33). Intranasal vaccination, when accompanied by IL-12 administration (which increases IFN-
production), resulted in increased serum IgG2a antipneumococcal antibody, consistent with the known stimulatory effect of IFN- on IgG2a production (34). This regimen also resulted in increased pneumococcal-specific IgA in BAL fluid. The serum antibody response was functional in opsonophagocytosis assays. Interestingly, intramuscular vaccination afforded more protection against subsequent intraperitoneal pneumococcal challenge compared with intranasal vaccination. In contrast, intranasal vaccination was more effective in reducing nasopharyngeal colonization after nasal challenge of the organism compared with intramuscular vaccination, despite the higher serum IgG response after the latter. Finally, using IgA knockout mice, these investigators demonstrated that IgA was responsible for reducing pneumococcal colonization after vaccination and intranasal challenge (33). These results again highlight the general principal that systemic immunization is more effective in providing protection against systemic challenges, whereas mucosal immunization is more protective against mucosal challenges.
In humans the pneumococcal vaccine is clearly effective in reducing the morbidity and mortality of invasive pneumococcal disease (35, 36). Despite this, studies describing the effect of vaccination on respiratory tract antibody concentrations are lacking. Virtually all studies examining the immunologic response to vaccination have measured plasma antibody levels. When reported, respiratory tract antibody measurements are usually done in saliva. Furthermore, most of these studies are in children where the burden of invasive pneumococcal disease is especially pronounced.
Several studies have examined systemic and salivary pneumococcal-specific antibody titers after systemic vaccination in children. Infants given the seven-valent conjugated pneumococcal vaccine at 2, 4, 6, and 15 mo had significant plasma IgG responses at 7 mo (37). Salivary pneumococcal-specific IgG and IgA concentrations were minimal at this time, however, and did not appear until a booster vaccine was given at 15 mo. Further studies suggested the salivary IgG was derived from serum, whereas the salivary IgA was locally produced. This same group demonstrated that by age 4 to 5 yr, antipneumococcal IgA, especially IgA1, was still present in saliva (38). Interestingly, there was no difference in pneumococcal-specific antibody titers between vaccinated and unvaccinated children at age 4 to 5 yr, suggesting that natural environmental exposure also resulted in mucosal immunity. Finally, immunization with the pneumococcal polysaccharide vaccine has been shown to lead to an increase in salivary pneumococcal-specific IgG and IgA in adults (39). In other studies, despite the lack of immunologic correlation, systemic pneumococcal vaccination seems to decrease the incidence of nasopharyngeal carriage of vaccine-specific serotypes (40).
In total, the studies described suggest systemic immunization can induce some local mucosal immune responses. The animal studies described in the beginning of this section, however, indicate that the strongest mucosal immune response is likely to be induced when antigen is introduced directly into the respiratory tract. Intranasal and inhaled vaccines offer advantages in addition to the potential for improved local immunity, including the ability to immunize large populations at less cost (41). In preliminary safety trials of intranasal vaccination against influenza using a virosome formulated inactivated virus the vaccine was associated with high serum concentrations of influenza-specific IgG and the presence of specific IgA in nasal lavage fluid (42). Similarly, an inhaled measles vaccine has been shown to be immunogenic (as determined by serum measurement of measles-specific IgG) in a large population of children (43). There is realistic hope for more immunogenic, more effective, and less expensive vaccination strategies against respiratory pathogens in the near future.
CONCLUSIONS
The humoral immune response is essential for host defense against bacteria. After exposure to potential pathogens, antibody responses in the respiratory tract can occur either quickly through activation of resident memory B cells if there has been prior bacterial exposure, or more slowly through the induction of both systemic and local mucosal immunity if the host is naive to the organism. The resulting production of antigen-specific IgG and IgA act in concert to help clear the invading pathogen and reduce subsequent colonization of respiratory epithelium. The type and concentration of antibody produced is dependent on the site of exposure (Figure 3). Upper airway exposure results in primarily an IgA response. Organisms that reach the lung after passing through the upper airway induce a more systemic response, including increased production of pathogen-specific IgG. Systemic vaccination against respiratory pathogens, although effective in generating systemic IgG responses and some mucosal IgA responses, may be less effective than vaccination through mucosal surfaces, which induce a brisk IgA and IgG response both locally and systemically depending on the site of vaccine deposition. Future studies should provide further insight on the pulmonary humoral host response to bacterial challenge and optimal vaccine regimens to minimize the burden of respiratory disease caused by pathogenic bacteria.
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Conflict of Interest Statement: H.L.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form August 10, 2005; accepted in final form August 18, 2005)
REFERENCES
This article has been cited by other articles:
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  M. Medina, J. Villena, E. Vintini, E. M. Hebert, R. Raya, and S. Alvarez Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice Infect. Immun., June 1, 2008; 76(6): 2696 - 2705. [Abstract] [Full Text] [PDF]
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0.05).