- Email: [email protected]
Effect of experimental influenza A infection on systemic immune and inflammatory parameters in allergic and nonallergic adult subjects
Effect of experimental influenza A infection on systemic immune and inflammatory parameters in allergic and nonallergic adult subjects Deborah A. Gentile, MD*; William J. Doyle, PhD†; Philip Fireman, MD*; and David P. Skoner, MD*†
Background: The economic impact and medical complication rate of viral upper respiratory infections are well documented, but many of the physiologic, inflammatory, and immune responses to respiratory viruses have only recently been investigated. A previous study demonstrated differential systemic immune and inflammatory responses in allergic rhinitis (AR) and nonallergic rhinitis (NAR) subjects during experimental infection with rhinovirus-39. Objective: The purpose of this study was to compare selected systemic immune and inflammatory responses to experimental influenza A virus (FLU) challenge in seronegative AR and NAR subjects. Methods: Peripheral blood was obtained at baseline (study day 0) and 3, 6, 18, and 31 days after intranasal FLU challenge and assayed for leukocyte histamine release, serum immunoglobulins, and plasma histamine. Results: All subjects were infected, as manifested by viral shedding in nasal secretions and/or seroconversion. FLU infection induced decreases in spontaneous leukocyte histamine release and increases in anti-immunoglobulin E–induced leukocyte histamine release, which were evident at least 1 month after infection, but caused no significant changes in serum immunoglobulins or plasma histamine. There were no differences between AR and NAR subjects for any of the study parameters. Conclusions: The results show that intranasal challenge with FLU induces changes in leukocyte histamine release, but not other systemic immune and inflammatory responses. Ann Allergy Asthma Immunol 2001;87:496–500.
INTRODUCTION Viral upper respiratory infections (URIs) are one of the most common reasons for physician’s office visits and school or work absenteeism. Although the economic impact and clinical complication rate of the common cold are well appreciated, researchers have only recently begun to identify the physiologic, inflammatory, and immune responses to respiratory virus* Departments of Pediatrics and † Otolaryngology, Children’s Hospital of Pittsburgh and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Supported by NIH grants NIH AI 19262 and 5MOI RR00084. Received for publication December 10, 2000. Accepted for publication in revised form August 28, 2001.
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es.1–7 Several problems have confounded the interpretation of the results of such studies, including the failure to vigorously define host characteristics, which might influence individual responsiveness to respiratory viruses. One such characteristic is the presence of upper airway allergy and inflammation, which could influence the local or systemic responses to respiratory viruses.7 In addition to the effects of allergy at the mucosal surface, abnormal function has been ascribed to a variety of immune and inflammatory cells in individuals with allergy, raising the possibility of abnormal responses to virus infection.8 –12 Indeed, a differential pattern of immune and inflammatory responses was observed in allergic rhinitis (AR) and
nonallergic rhinitis (NAR) subjects experimentally infected with rhinovirus-39 (RV-39).13,14 In those studies, acute increases in serum immunoglobulin (Ig)E levels were confined to allergic subjects and acute increases in leukocyte histamine release and platelet aggregation were greater in allergic as compared with nonallergic subjects. The purpose of the current study, conducted in conjunction with protocols which measured nasal physiology,15 was 2-fold: to monitor selected systemic immune and inflammatory parameters, including serum IgE levels, plasma histamine levels, and leukocyte histamine release, during experimental FLU infection, and to compare these responses in AR and NAR subjects. METHODS Study Population Adult subjects were recruited from the University of Pittsburgh and the surrounding community. All were in good health as evidenced by medical history, physical examination, normal blood chemistry and hematology profiles, normal urine examination, and negative HIV antibody assay. None of the subjects experienced a symptomatic URI within the 30 days before the study and all had a pre-inoculation hemagglutination-inhibition (HI) serum antibody titer to the challenge virus of ⱕ10. Twenty-seven subjects (8 male, 19 female, 18 to 54 years old), with (n ⫽ 12) or without (n ⫽ 15) allergic rhinitis,15 were enrolled after providing written informed consent. The study was approved by the Human Rights Committee at Children’s Hospital of Pittsburgh.
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Study Protocol The study methods used for FLU inoculation and collection of physiologic and symptomatic data have been described previously.15 Briefly, enrolled subjects were cloistered in individual rooms of a local hotel for an 8-day period (study days 0 to 7). Twentyfour hours after admission to the cloister site (at the end of study day 0), the subjects were intranasally inoculated with 107 median tissue culture infective dose of a safety-tested clinical isolate of influenza A/Kawasaki (H1N1) supplied by the National Institute of Allergy and Infectious Diseases (wild type; lot E-262). During the morning of each study day, subjects were given a physical examination, vital signs were recorded, and a nasal lavage was performed for viral culture. On a daily basis, symptom scores were evaluated, nasal secretion weights were calculated, and a battery of physiologic tests was completed. Subjects were dismissed from cloister on the afternoon of study day 8. During the study, all subjects were asked to refrain from taking medications, with the exception of oral contraceptives. Peripheral blood was obtained at baseline (study day 0) and 3, 6, 18, and 31 days after intranasal FLU challenge. All blood samples were obtained at approximately the same time each morning (7:30 to 9:30 AM) and delivered immediately to the laboratory for processing. Spontaneous and anti-IgE–induced leukocyte histamine release, plasma histamine levels, and serum total IgG, IgA, IgM, and IgE levels were evaluated on each of these days. Convalescent serum HI titers to the challenge virus were evaluated on study day 31. Methods for Evaluating Viral Infection The methods for performing nasal lavage, culturing FLU from nasal lavage samples, and assaying serum HI titers to the challenge virus were described previously.15 A subject was considered to be infected with FLU if the challenge virus was isolated from the lavage fluids on at least 1 postchallenge
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day of cloister or if a ⱖ 4-fold increase in serum-specific HI antibody titer between pre-inoculation and convalescent samples was documented. Leukocyte Histamine Release Leukocyte histamine release to 100 g/mL anti-IgE antibody (Cappel, Research Triangle Park, NC) was assayed on blood samples using the whole blood assay previously described.16 Histamine was assayed by radioimmunoassay (AMAC, Inc, Westbrook, ME) and results were expressed as a percentage of total histamine release. Spontaneous (non-induced) histamine release was always ⬍10% of the total histamine content. Plasma Histamine Levels Blood was atraumatically collected into chilled vacutainer collection tubes containing disodium ethylenediaminetetraacetic acid. After centrifugation at 4° C, the top half of the plasma layer was pipetted into polypropylene tubes for storage at ⫺70° C until histamine was assayed by radioimmunoassay (AMAC, Inc).17 Serum Ig Levels Total serum IgE levels were determined by the fluoroallergosorbent test (FAST; Biowhitaker, Inc, Walkersville, MD).18 Serum levels of IgG, IgA, and IgM were determined by nephelometry (Sanofi Diagnostics Pasteur, Inc, Chaska, MN).19 Statistical Methods Outcome variables for this study consisted of the plasma histamine levels, the serum concentrations of the immunoglobulins (IgG, IgA, IgM, and IgE), and spontaneous and anti-IgE–induced leukocyte histamine release on days 0, 3, 6, 18, and 31 for subjects in the AR and NAR subgroups. The primary analysis was done on each variable using a repeated measures analysis of variance (ANOVA), with variance partitioned by subgroup and study day. Where significant effects of study day were documented, pairwise differences between postexposure daily values and the pre-exposure baseline values were evaluated for significance using a
paired Student’s t test evaluated at ␣P ⬍ 0.03 to control for multiple comparisons. Unless otherwise specified, the format mean ⫾ standard error of the mean is used to express summary data. All statistical procedures were run using the NCSS 2000 Statistical Package (Kaysville, UT). RESULTS Infection and Illness A panel of descriptive clinical and laboratory parameters related to the FLU challenge was presented previously.15 All subjects were infected as evidenced by virus shedding or seroconversion. The peak in nasal symptoms occurred at day 4 with the AR subgroup having a greater magnitude. The peak in throat and systemic symptom scores occurred at day 2 with the AR subgroup having a greater magnitude for throat symptom scores. Secretion weights peaked at day 3 for the AR subgroup and at day 4 for the NAR subgroup. All symptoms and signs showed a progressive decrease to approximate baseline levels by study day 7. Plasma Histamine Levels The average baseline plasma histamine concentrations (ng/mL) for the NAR and AR subgroups was 0.18 ⫾ 0.12 and 0.21 ⫾ 0.10, respectively. There were no significant effects by ANOVA of either study day (max NAR ⫽ 0.18 ⫾ 0.08 on day 3; max AR ⫽ 0.23 ⫾ 0.09 on day 18) or subgroup assignment on that variable. Leukocyte Histamine Release ANOVA documented a significant effect of study day but not subgroup assignment on both the spontaneous (P ⬍ 0.001) and anti-IgE–induced (P ⬍ 0.02) leukocyte histamine release. With the exception of the baseline versus day 18 comparison for spontaneous leukocyte histamine release, all pairwise comparisons of the values recorded on the postexposure study days with the baseline values were statistically significant. Figure 1 shows the average spontaneous (A) and anti-IgE–induced (B) percentage of leukocyte histamine release as a function of study day for each subgroup.
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Figure 1. Average spontaneous (A) and anti-IgE–induced (B) leukocyte histamine release (% total) for AR (f) and NAR (F) subjects as a function of study day. Horizontal bars demarcate the bounds of the standard error of the mean.
Serum Ig Levels For the NAR and AR subgroups, average baseline values (IU/mL) of IgG were 1,089 ⫾ 159 and 1,035 ⫾ 179; of IgA were 232 ⫾ 83 and 220 ⫾ 67; of IgM were 115 ⫾ 46 and 110 ⫾ 45; and of IgE were 9 ⫾ 9 and 82 ⫾ 107, respectively. ANOVA documented a significant difference between groups at baseline for IgE (P ⬍ 0.02) but not the other immunoglobulins; however, there were no significant changes in IgE levels within either subgroup at any time point after inoculation. Figure 2 shows the average serum IgE levels as a function of study day for each subgroup. DISCUSSION The results of this study show that intranasal challenge with FLU induced significant decreases in spontaneous leukocyte histamine release and increases in anti-IgE–induced leukocyte histamine release, which were evident at least 1 month after infection, but caused no significant changes in
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plasma histamine or total serum IgG, IgA, IgM, or IgE levels. Further, the alterations in leukocyte histamine release were observed in both AR and NAR subjects. The observation that serum IgG, IgA, and IgM levels were not changed by experimental FLU infection was expected and consistent with results from a previous study examining the effect
of experimental RV-39 infection on these responses. However, it is somewhat surprising that AR subjects did not manifest increased serum IgE levels during experimental FLU infection. AR subjects have a well documented propensity to overproduce IgE, as evidenced by previous literature and as manifested by subjects at baseline in the present study.12,20 Further increases in serum IgE levels have been observed in association with seasonal allergen exposure and a variety of viral infections, including natural infection with Epstein-Barr virus, cytomegalovirus, and respiratory syncytial, influenza, parainfluenza, adenovirus, and herpes simplex viruses.21–23 Similarly, our laboratory previously reported increases in serum IgE levels during experimental infection with RV-39.14 That study used the same personnel and cloister site as this study. However, the acute IgE elevations observed in previous natural and experimental viral studies were modest compared with the approximately 2- to 4-fold increases observed during natural pollen exposure in pollen-sensitive individuals. The results of the present study do not rule out the possibility that AR subjects manifest increased levels of specific IgE against the study virus. Indeed, previous studies have reported elevations of specific IgE levels against respiratory syncytial virus in children naturally infected with this virus.24,25 Experimental FLU infection significantly decreased spontaneous leuko-
Figure 2. Average IgE concentrations for AR (f) and NAR (F) subjects as a function of study day. Horizontal bars demarcate the bounds of the standard error of the mean.
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cyte histamine release; however, the magnitude of this effect was relatively small and unlikely to be of clinical relevance. The observation that experimental FLU infection induced acute increases in anti-IgE–induced leukocyte histamine release is consistent with the results of studies using in vitro virus infection of mediator releasing cells and may be attributable to an enhanced production of a histaminereleasing factor by other cell types.26,27 It is conceivable that this heightened response was IgE-mediated; however, the lack of significant elevations in serum levels of this antibody during this study does not support this hypothesis. Other studies reported increased leukocyte histamine release during experimental infection with RV-16 and -39; however, in those studies, the response was confined only to AR subjects.28 Despite the enhanced anti-IgE–induced leukocyte histamine release, there was no concomitant elevation in plasma histamine levels during the FLU infection. The lack of histamine elevations in peripheral blood suggests at least two possibilities. First, the blood sampling frequency selected in the current study was not often enough to detect this rapidly metabolized mediator. This hypothesis is supported by several studies in which local nasal lavage levels of histamine and urinary levels of histamine metabolites were elevated in subjects infected with RV-39 and FLU.29,30 However, this hypothesis is less likely given the fact that the levels observed in this study are consistent with normal plasma levels reported in other studies.31,32 The second possibility is that histamine may not be released in significant quantities during FLU infection. Support of this hypothesis has been forthcoming from investigators using models of both experimental and naturally acquired infections.33,34 CONCLUSION In the present study, AR (as compared with NAR) subjects did not manifest enhanced systemic immune and inflammatory responses during experimental FLU infection. This may be
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attributable to a variety of factors. For example, in this study, the provoked illness was relatively mild and there was no concomitant exposure to other factors known to modulate these responses (eg, allergen). Additionally, the sample size was relatively small and may have resulted in a type 2 error. Alternatively, AR (as compared with NAR) subjects may not manifest enhanced systemic immune and inflammatory responses during FLU infection. In support of this hypothesis, several recent studies failed to document enhanced illness or immune and inflammatory responses in allergenprimed AR (as compared with NAR) subjects during experimental URIs.35,36 It is important to note that the results of the present study do not role out the possibility that AR subjects manifest enhanced local immune and inflammatory responses during FLU infection. Future studies need to examine this issue.
8. 9.
10.
11.
12.
REFERENCES 1. Doyle WJ, Skoner DP, Fireman P, et al. Rhinovirus 39 infection in allergic and nonallergic subjects. J Allergy Clin Immunol 1992;89:968 –978. 2. Igarashi Y, Skoner DP, Doyle WJ, et al. Analysis of nasal secretions during experimental rhinovirus upper respiratory infections. J Allergy Clin Immunol 1993;93:722–731. 3. Hsia J, Goldstein AL, Simon GL, et al. Peripheral blood mononuclear cell interleukin-2 and interferon-␥ production, cytotoxicity, and antigen-stimulated blastogenesis during experimental rhinovirus infection. J Infect Dis 1990;162:591–597. 4. Hsia J, Sztein MB, Naylor PH, et al. Modulation of thymosin ␣1 and thymosin 4 levels and peripheral blood mononuclear cell subsets during experimental rhinovirus colds. Lymphokine Res 1989;8:383–391. 5. Levandowski RA, Ou DW, Jackson GG. Acute-phase decrease of T lymphocyte subsets in rhinovirus infection. J Infect Dis 1986;153:743–748. 6. Levandowski RA, Weaver CW, Jackson GG. Nasal-secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection. J Med Virol 1988;25:423– 432. 7. Calhoun WJ, Swenson CA, Dick EC, et al. Experimental rhinovirus 16 in-
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fection potentiates histamine release after antigen bronchoprovocation in allergic subjects. Am Rev Respir Dis 1991;144:1267–1273. Timonen T, Stenius-Aarniala B. Natural killer cell activity in asthma. Clin Exp Immunol 1985;59:85–90. Vesterinen E, Timonen T. Natural killer cell activity in specific and nonspecific bronchial challenge. Ann Allergy 1988;60:247–249. Maccia CA, Gallagher SJ, Ataman G, et al. Platelet thrombopathy in asthmatic patients with elevated immunoglobulin E. J Allergy Clin Immunol 1977;59:101–108. Makino S, Ikemori K, Kashima T, Fukada T. Comparison of cyclic adenosine monophosphate response of lymphocytes in normal and asthmatic subjects to norepinephrine and salbutamol. J Allergy Clin Immunol 1977;59:348 –352. Weller FR, Kallenberg CG, Jansen HM, et al. The primary immune response in bronchial asthma. I. A kinetic study of Helix pomatia hemocyanin-specific IgE, IgG, IgA, and IgM antibody responses in patients with asthma and in matched controls. J Allergy Clin Immunol 1985;76:29 –34. Skoner DP, Whiteside TL, Wilson JW, et al. Effect of rhinovirus 39 infection on cellular immune parameters in allergic and nonallergic subjects. J Allergy Clin Immunol 1993;92:732–743. Skoner DP, Doyle WJ, Tanner EP, et al. Effect of rhinovirus 39 (RV-39) infection on immune and inflammatory parameters in allergic and non-allergic subjects. Clin Exp Allergy 1995;25: 561–567. Doyle WJ, Skoner DP, Hayden F, et al. Nasal and otologic effects of experimental influenza A virus infection. Ann Otol Rhinol Laryngol 1994;103: 59 – 69. Lorenz W, Reimann HJ, Barth H, et al. A sensitive and specific method for the determination of histamine in human whole blood and plasma. Koppe Seylers Z Physiol Chem 1972;355: 911–920. Skoner DP, Page R, Asman B, et al. Plasma elevations of histamine and a prostaglandin metabolite in acute asthma. Am Rev Respir Dis 1988;137: 1009 –1014. Tsay YG, Halpern GM. Fluorescent enzyme immunoassay for serum IgE (total IgE FAST). In: Rapid Methods
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and Automation in Microbiology and Immunology. Fourth International Symposium. Springer-Verlag: Berlin.1985;20. Sternberg JC. Immunogloblin quantitation. In: Rose NR, Friedman H, Fahey JL, editors. Manual of Clinical Laboratory Immunology. 3rd ed. Washington, DC: American Society for Microbiology, 1986. Hemady Z, Blomberg F, Gellis S, Rocklin RE. IgE production in vitro by human blood mononuclear cells: a comparison between atopic and nonatopic subjects. J Allergy Clin Immunol 1983;71:324 –330. Bahna SL, Horwitz CA, Fiala M, Heiner DC. IgE response in heterophilpositive infectious mononucleosis. J Allergy Clin Immunol 1978;62: 167–173. Yunginger JW, Gleich GJ. Seasonal changes in serum and nasal IgE concentrations. J Allergy Clin Immunol 1973;51:174 –186. Polmar SH, Robinson LD Jr, Minnefor AB. Immunoglobulin E in bronchiolitis. Pediatrics 1972;50:279 –284. Welliver RC, Wong DT, Sun M, et al. The development of respiratory syncytial virus-specific IgE and release of histamine in nasopharyngeal secretions after infection. N Engl J Med 1981;305:841– 846. Welliver RC, Duffy L. The relationship of RSV-specific immunoglobulin
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E antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7– 8 years. Pediatr Pulmonol 1993;15:19 –27. Busse WW, Swenson CA, Borden EC, et al. Effect of influenza A virus on leukocyte histamine release. J Allergy Clin Immunol 1983;71:382–388. Chonmaitree T, Lett-Brown MA, Grant JA. Respiratory viruses induce production of histamine-releasing factor by mononuclear leukocytes: a possible role in the mechanism of virusinduced asthma. J Infect Dis 1991;164: 592–594. Lemanske RF Jr, Dick EC, Swenson CA, et al. Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J Clin Invest 1989;83:1–10. Igarashi Y, Skoner DP, Doyle WJ, et al. Analysis of nasal secretions during experimental rhinovirus upper respiratory infections. J Allergy Clin Immunol 1993;92:722–731. Skoner DP, Fireman P, Doyle WJ. Urine histamine metabolite elevations during experimental colds. J Allergy Clin Immunol 1997;99:S419. Lin RY, Schwartz LB, Curry A, et al. Histamine and tryptase levels in patients with acute allergic reactions: an emergency department-based study. J Allergy Clin Immunol 2000;106: 65–71. Ohtusuka T, Matsumaru S, Uchida K,
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et al. Time course of plasma histamine and tryptase following food challenges in children with suspected food allergy. Ann Allergy 1993;71:139 –146. Naclerio RM, Proud D, Kagey-Sobotka A, et al. Is histamine responsible for the symptoms of rhinovirus colds? A look at the inflammatory mediators following infection. Pediatr Infect Dis J 1988;7:218 –222. Berkowitz RB, Tinkelman DG. Evaluation of oral terfenadine for treatment of the common cold. Ann Allergy 1991;67:593–597. Exelbert RL, Skoner DP, Gentile DA, et al. Upper airway responses in ragweed-primed and unprimed adult subjects during experimental infection with influenza A virus. J Allergy Clin Immunol 2000;105:S198. Avila PC, Abisheganaden JA, Wong H, et al. Effects of allergic inflammation of the nasal mucosa on the severity of rhinovirus 16 cold. J Allergy Clin Immunol 2000;105:923–932.
Requests for reprints should be addressed to: Deborah A. Gentile, MD Section of Allergy/Immunology Children’s Hospital of Pittsburgh 3705 Fifth Avenue Pittsburgh, PA 15213 E-mail: [email protected]
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Background: The economic impact and medical complication rate of viral upper respiratory infections are well documented, but many of the physiologic, inflammatory, and immune responses to respiratory viruses have only recently been investigated. A previous study demonstrated differential systemic immune and inflammatory responses in allergic rhinitis (AR) and nonallergic rhinitis (NAR) subjects during experimental infection with rhinovirus-39. Objective: The purpose of this study was to compare selected systemic immune and inflammatory responses to experimental influenza A virus (FLU) challenge in seronegative AR and NAR subjects. Methods: Peripheral blood was obtained at baseline (study day 0) and 3, 6, 18, and 31 days after intranasal FLU challenge and assayed for leukocyte histamine release, serum immunoglobulins, and plasma histamine. Results: All subjects were infected, as manifested by viral shedding in nasal secretions and/or seroconversion. FLU infection induced decreases in spontaneous leukocyte histamine release and increases in anti-immunoglobulin E–induced leukocyte histamine release, which were evident at least 1 month after infection, but caused no significant changes in serum immunoglobulins or plasma histamine. There were no differences between AR and NAR subjects for any of the study parameters. Conclusions: The results show that intranasal challenge with FLU induces changes in leukocyte histamine release, but not other systemic immune and inflammatory responses. Ann Allergy Asthma Immunol 2001;87:496–500.
INTRODUCTION Viral upper respiratory infections (URIs) are one of the most common reasons for physician’s office visits and school or work absenteeism. Although the economic impact and clinical complication rate of the common cold are well appreciated, researchers have only recently begun to identify the physiologic, inflammatory, and immune responses to respiratory virus* Departments of Pediatrics and † Otolaryngology, Children’s Hospital of Pittsburgh and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Supported by NIH grants NIH AI 19262 and 5MOI RR00084. Received for publication December 10, 2000. Accepted for publication in revised form August 28, 2001.
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es.1–7 Several problems have confounded the interpretation of the results of such studies, including the failure to vigorously define host characteristics, which might influence individual responsiveness to respiratory viruses. One such characteristic is the presence of upper airway allergy and inflammation, which could influence the local or systemic responses to respiratory viruses.7 In addition to the effects of allergy at the mucosal surface, abnormal function has been ascribed to a variety of immune and inflammatory cells in individuals with allergy, raising the possibility of abnormal responses to virus infection.8 –12 Indeed, a differential pattern of immune and inflammatory responses was observed in allergic rhinitis (AR) and
nonallergic rhinitis (NAR) subjects experimentally infected with rhinovirus-39 (RV-39).13,14 In those studies, acute increases in serum immunoglobulin (Ig)E levels were confined to allergic subjects and acute increases in leukocyte histamine release and platelet aggregation were greater in allergic as compared with nonallergic subjects. The purpose of the current study, conducted in conjunction with protocols which measured nasal physiology,15 was 2-fold: to monitor selected systemic immune and inflammatory parameters, including serum IgE levels, plasma histamine levels, and leukocyte histamine release, during experimental FLU infection, and to compare these responses in AR and NAR subjects. METHODS Study Population Adult subjects were recruited from the University of Pittsburgh and the surrounding community. All were in good health as evidenced by medical history, physical examination, normal blood chemistry and hematology profiles, normal urine examination, and negative HIV antibody assay. None of the subjects experienced a symptomatic URI within the 30 days before the study and all had a pre-inoculation hemagglutination-inhibition (HI) serum antibody titer to the challenge virus of ⱕ10. Twenty-seven subjects (8 male, 19 female, 18 to 54 years old), with (n ⫽ 12) or without (n ⫽ 15) allergic rhinitis,15 were enrolled after providing written informed consent. The study was approved by the Human Rights Committee at Children’s Hospital of Pittsburgh.
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
Study Protocol The study methods used for FLU inoculation and collection of physiologic and symptomatic data have been described previously.15 Briefly, enrolled subjects were cloistered in individual rooms of a local hotel for an 8-day period (study days 0 to 7). Twentyfour hours after admission to the cloister site (at the end of study day 0), the subjects were intranasally inoculated with 107 median tissue culture infective dose of a safety-tested clinical isolate of influenza A/Kawasaki (H1N1) supplied by the National Institute of Allergy and Infectious Diseases (wild type; lot E-262). During the morning of each study day, subjects were given a physical examination, vital signs were recorded, and a nasal lavage was performed for viral culture. On a daily basis, symptom scores were evaluated, nasal secretion weights were calculated, and a battery of physiologic tests was completed. Subjects were dismissed from cloister on the afternoon of study day 8. During the study, all subjects were asked to refrain from taking medications, with the exception of oral contraceptives. Peripheral blood was obtained at baseline (study day 0) and 3, 6, 18, and 31 days after intranasal FLU challenge. All blood samples were obtained at approximately the same time each morning (7:30 to 9:30 AM) and delivered immediately to the laboratory for processing. Spontaneous and anti-IgE–induced leukocyte histamine release, plasma histamine levels, and serum total IgG, IgA, IgM, and IgE levels were evaluated on each of these days. Convalescent serum HI titers to the challenge virus were evaluated on study day 31. Methods for Evaluating Viral Infection The methods for performing nasal lavage, culturing FLU from nasal lavage samples, and assaying serum HI titers to the challenge virus were described previously.15 A subject was considered to be infected with FLU if the challenge virus was isolated from the lavage fluids on at least 1 postchallenge
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day of cloister or if a ⱖ 4-fold increase in serum-specific HI antibody titer between pre-inoculation and convalescent samples was documented. Leukocyte Histamine Release Leukocyte histamine release to 100 g/mL anti-IgE antibody (Cappel, Research Triangle Park, NC) was assayed on blood samples using the whole blood assay previously described.16 Histamine was assayed by radioimmunoassay (AMAC, Inc, Westbrook, ME) and results were expressed as a percentage of total histamine release. Spontaneous (non-induced) histamine release was always ⬍10% of the total histamine content. Plasma Histamine Levels Blood was atraumatically collected into chilled vacutainer collection tubes containing disodium ethylenediaminetetraacetic acid. After centrifugation at 4° C, the top half of the plasma layer was pipetted into polypropylene tubes for storage at ⫺70° C until histamine was assayed by radioimmunoassay (AMAC, Inc).17 Serum Ig Levels Total serum IgE levels were determined by the fluoroallergosorbent test (FAST; Biowhitaker, Inc, Walkersville, MD).18 Serum levels of IgG, IgA, and IgM were determined by nephelometry (Sanofi Diagnostics Pasteur, Inc, Chaska, MN).19 Statistical Methods Outcome variables for this study consisted of the plasma histamine levels, the serum concentrations of the immunoglobulins (IgG, IgA, IgM, and IgE), and spontaneous and anti-IgE–induced leukocyte histamine release on days 0, 3, 6, 18, and 31 for subjects in the AR and NAR subgroups. The primary analysis was done on each variable using a repeated measures analysis of variance (ANOVA), with variance partitioned by subgroup and study day. Where significant effects of study day were documented, pairwise differences between postexposure daily values and the pre-exposure baseline values were evaluated for significance using a
paired Student’s t test evaluated at ␣P ⬍ 0.03 to control for multiple comparisons. Unless otherwise specified, the format mean ⫾ standard error of the mean is used to express summary data. All statistical procedures were run using the NCSS 2000 Statistical Package (Kaysville, UT). RESULTS Infection and Illness A panel of descriptive clinical and laboratory parameters related to the FLU challenge was presented previously.15 All subjects were infected as evidenced by virus shedding or seroconversion. The peak in nasal symptoms occurred at day 4 with the AR subgroup having a greater magnitude. The peak in throat and systemic symptom scores occurred at day 2 with the AR subgroup having a greater magnitude for throat symptom scores. Secretion weights peaked at day 3 for the AR subgroup and at day 4 for the NAR subgroup. All symptoms and signs showed a progressive decrease to approximate baseline levels by study day 7. Plasma Histamine Levels The average baseline plasma histamine concentrations (ng/mL) for the NAR and AR subgroups was 0.18 ⫾ 0.12 and 0.21 ⫾ 0.10, respectively. There were no significant effects by ANOVA of either study day (max NAR ⫽ 0.18 ⫾ 0.08 on day 3; max AR ⫽ 0.23 ⫾ 0.09 on day 18) or subgroup assignment on that variable. Leukocyte Histamine Release ANOVA documented a significant effect of study day but not subgroup assignment on both the spontaneous (P ⬍ 0.001) and anti-IgE–induced (P ⬍ 0.02) leukocyte histamine release. With the exception of the baseline versus day 18 comparison for spontaneous leukocyte histamine release, all pairwise comparisons of the values recorded on the postexposure study days with the baseline values were statistically significant. Figure 1 shows the average spontaneous (A) and anti-IgE–induced (B) percentage of leukocyte histamine release as a function of study day for each subgroup.
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Figure 1. Average spontaneous (A) and anti-IgE–induced (B) leukocyte histamine release (% total) for AR (f) and NAR (F) subjects as a function of study day. Horizontal bars demarcate the bounds of the standard error of the mean.
Serum Ig Levels For the NAR and AR subgroups, average baseline values (IU/mL) of IgG were 1,089 ⫾ 159 and 1,035 ⫾ 179; of IgA were 232 ⫾ 83 and 220 ⫾ 67; of IgM were 115 ⫾ 46 and 110 ⫾ 45; and of IgE were 9 ⫾ 9 and 82 ⫾ 107, respectively. ANOVA documented a significant difference between groups at baseline for IgE (P ⬍ 0.02) but not the other immunoglobulins; however, there were no significant changes in IgE levels within either subgroup at any time point after inoculation. Figure 2 shows the average serum IgE levels as a function of study day for each subgroup. DISCUSSION The results of this study show that intranasal challenge with FLU induced significant decreases in spontaneous leukocyte histamine release and increases in anti-IgE–induced leukocyte histamine release, which were evident at least 1 month after infection, but caused no significant changes in
498
plasma histamine or total serum IgG, IgA, IgM, or IgE levels. Further, the alterations in leukocyte histamine release were observed in both AR and NAR subjects. The observation that serum IgG, IgA, and IgM levels were not changed by experimental FLU infection was expected and consistent with results from a previous study examining the effect
of experimental RV-39 infection on these responses. However, it is somewhat surprising that AR subjects did not manifest increased serum IgE levels during experimental FLU infection. AR subjects have a well documented propensity to overproduce IgE, as evidenced by previous literature and as manifested by subjects at baseline in the present study.12,20 Further increases in serum IgE levels have been observed in association with seasonal allergen exposure and a variety of viral infections, including natural infection with Epstein-Barr virus, cytomegalovirus, and respiratory syncytial, influenza, parainfluenza, adenovirus, and herpes simplex viruses.21–23 Similarly, our laboratory previously reported increases in serum IgE levels during experimental infection with RV-39.14 That study used the same personnel and cloister site as this study. However, the acute IgE elevations observed in previous natural and experimental viral studies were modest compared with the approximately 2- to 4-fold increases observed during natural pollen exposure in pollen-sensitive individuals. The results of the present study do not rule out the possibility that AR subjects manifest increased levels of specific IgE against the study virus. Indeed, previous studies have reported elevations of specific IgE levels against respiratory syncytial virus in children naturally infected with this virus.24,25 Experimental FLU infection significantly decreased spontaneous leuko-
Figure 2. Average IgE concentrations for AR (f) and NAR (F) subjects as a function of study day. Horizontal bars demarcate the bounds of the standard error of the mean.
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cyte histamine release; however, the magnitude of this effect was relatively small and unlikely to be of clinical relevance. The observation that experimental FLU infection induced acute increases in anti-IgE–induced leukocyte histamine release is consistent with the results of studies using in vitro virus infection of mediator releasing cells and may be attributable to an enhanced production of a histaminereleasing factor by other cell types.26,27 It is conceivable that this heightened response was IgE-mediated; however, the lack of significant elevations in serum levels of this antibody during this study does not support this hypothesis. Other studies reported increased leukocyte histamine release during experimental infection with RV-16 and -39; however, in those studies, the response was confined only to AR subjects.28 Despite the enhanced anti-IgE–induced leukocyte histamine release, there was no concomitant elevation in plasma histamine levels during the FLU infection. The lack of histamine elevations in peripheral blood suggests at least two possibilities. First, the blood sampling frequency selected in the current study was not often enough to detect this rapidly metabolized mediator. This hypothesis is supported by several studies in which local nasal lavage levels of histamine and urinary levels of histamine metabolites were elevated in subjects infected with RV-39 and FLU.29,30 However, this hypothesis is less likely given the fact that the levels observed in this study are consistent with normal plasma levels reported in other studies.31,32 The second possibility is that histamine may not be released in significant quantities during FLU infection. Support of this hypothesis has been forthcoming from investigators using models of both experimental and naturally acquired infections.33,34 CONCLUSION In the present study, AR (as compared with NAR) subjects did not manifest enhanced systemic immune and inflammatory responses during experimental FLU infection. This may be
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attributable to a variety of factors. For example, in this study, the provoked illness was relatively mild and there was no concomitant exposure to other factors known to modulate these responses (eg, allergen). Additionally, the sample size was relatively small and may have resulted in a type 2 error. Alternatively, AR (as compared with NAR) subjects may not manifest enhanced systemic immune and inflammatory responses during FLU infection. In support of this hypothesis, several recent studies failed to document enhanced illness or immune and inflammatory responses in allergenprimed AR (as compared with NAR) subjects during experimental URIs.35,36 It is important to note that the results of the present study do not role out the possibility that AR subjects manifest enhanced local immune and inflammatory responses during FLU infection. Future studies need to examine this issue.
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Requests for reprints should be addressed to: Deborah A. Gentile, MD Section of Allergy/Immunology Children’s Hospital of Pittsburgh 3705 Fifth Avenue Pittsburgh, PA 15213 E-mail: [email protected]
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