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Viral infections have been associated with cellular immune responses and production of Th-1 cytokines. Respiratory syncytial virus (RSV), however, induces virus-specific IgE, which might be a consequence of a Th-2-like activation. To test this hypothesis we quantified interferon-
(IFN-) and interleukin-4 (IL-4) in the supernatant of peripheral blood mononuclear cells cultured for 24 and 48 h in
the presence or absence of phytohemaglutinin and pokeweed mitogen and the lymphocyte phenotypes to analyze subsets and their activation markers, from 15 hospitalized infants during an acute
lower respiratory infection caused by RSV and 17 healthy control infants from 1 to 15 mo of age.
Compared with the control infants, those infected with RSV had an increase in the number of B-cells
(p < 0.02) and decreases in both CD8+ T-cells (p < 0.01) and activated CD8+/CD25+ suppressor/
cytotoxic T-cells (p < 0.007). In RSV-infected infants, IFN- production was subtotally suppressed,
whereas IL-4 production was decreased to a lesser degree, giving significantly (p < 0.001) increased
IL-4/IFN- ratio compared with that in the control infants. These findings suggest a predominant
Th-2-like response in RSV-infected infants, which could explain some aspects of the immunopathogenesis of RSV infection and the RSV-specific and nonspecific IgE antibody responses observed.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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Respiratory syncytial virus (RSV) is one of the principal causes of morbidity and mortality worldwide (1). The immunology and pathogenesis of RSV is not fully understood. Children with cellular immunodeficiency have severe infections and impaired capacity to eliminate the virus. The disease is most common during the first 6 mo of life despite higher levels of maternally transmitted serum antibody (2, 3). This observation suggest that cellular immunity is important in host defense against RSV.
Cellular immunity has classically been described as the defense mechanism for viral infections and other intracellular
pathogens. The development of cellular or humoral immune
responses will depend on a repertoire of cytokines produced
by numerous cells, including CD4+ helper T-cells. These lymphocytes can be subdivided into two subsets, Th-1 and Th-2,
on the basis of the cytokine profile (4). Th-1 cells are characterized by secretion of interferon- (IFN-) and interleukin-2
(IL-2), and promote cell-mediated immunity. Th-2 cells selectively produce IL-4 and IL-5, and they are involved in the development of humoral immunity. Each Th cell subset inhibits its counterpart either directly or indirectly. Th-2 cells have a
prominent role in immediate-type hypersensitivity, as IL-4 is the critical stimulus inducing the isotype switch to IgE antibody production, whereas IFN- inhibits the switch (5). IFN-
has direct antiviral activity; it also has an indirect effect by
stimulating the cytolitic activity of natural killer (NK) cells
and CD8+ T-cells. RSV infection results in relative low levels
of IFN synthesis compared with other viruses (6). A cell-mediated cytotoxic response to RSV has been demonstrated in the
peripheral blood of only four of 22 infants with acute RSV
bronchiolitis (7). This cellular immune response was found in
infants with mild infection but not in those with the most severe infection, which suggests that the cellular immune response may not be primarily involved in the pathogenesis of
RSV infection.
In a certain percentage of children with atopic genetic predisposition, RSV infection has been related to the development of recurrent episodes of bronchial obstruction, established asthma, and/or allergies (8). The precise mechanism by which RSV can induce allergies and asthma has not been elucidated. However, one possible pathogenic mechanism could be through increasing the synthesis of IgE since it has been demonstrated that RSV can induce an IgE antivirus response (11, 12), and that RSV-specific IgE responses in infancy are associated with later recurrence of wheezing (13). Of note, the mechanism by which RSV can induce IgE synthesis is not known. Using mice vaccinated with recombinant vaccinia viruses, to express specific RSV proteins, it was found that the protein-G-specific T-cells had a Th-2-type cytokine profile, with large amounts of IL-4 and IL-5 production. In contrast, fusion protein-specific T-cells consisted of a mixture of CD8+ and Th-1-type CD4+ cells, suggesting that the type of immunologic response may depend on the processed RSV protein (14, 15).
The antecedents suggest that RSV infections stimulate a poor cellular immune response and that the pathogenesis could be related to an inflammatory response mediated by cytokines of the Th-2 profile that promote IgE production.
The aim of this work was to study the type of immune response (Th-1 or Th-2) during an episode of acute lower respiratory infection (ALRI) caused by RSV. To perform the study
we quantified IFN- and IL-4 in the supernatant of phytohemaglutinin (PHA) and pokeweed mitogen (PWM) cultured
peripheral blood mononuclear cells (PBMC) and the lymphocytes phenotype to analyzed lymphocyte subsets and their activation markers.
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METHODS
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DISCUSSION
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Reagents
Sodium metrizoate (Lymphoprep; Nycomed Pharma AS, Oslo, Norway), AIM-V culture medium (GIBCO, BRL, Grand Island, NY), monoclonal antibodies (Becton-Dickinson Immunocytometry Systems, San José, CA) to CD45, CD3, CD4, CD8, CD19, CD14, CD25, HLADR, and goat antimouse IgG directly conjugated to fluorescein isothyiocyanate or phycoerythrin, and mouse monoclonal antibody to respiratory syncytial virus (Centers for Disease Control, Atlanta, GA) were obtained. Phytohemaglutinin and pokeweed mitogen were purchased from Sigma (St. Louis, MO).
Selection of Patients
Fifteen infants between 1 and 13 mo of age (median, 6 mo), admitted for ALRI to Roberto del Río Children's hospital in Santiago, Chile, during the epidemic period of 1992 to 1994, were enrolled. They were healthy until the time of RSV infection, and all had a severe infection with bronchial obstruction with or without bronchopneumonia. The infected group was further divided into those with and those without a family history of atopic disease. The history of atopy of parents, grandparents, and siblings was performed by a questionnaire considering symptoms, laboratory tests, diagnosis by a specialist, and treatment for bronchial asthma and/or allergic rhinitis. A healthy control group of 17 infants between 1 and 16 mo of age (median, 8 mo) who required blood tests prior to minor surgery, was selected from the outpatient otorhinolaryngology or ophthalmology clinics. Nasopharyngeal aspirates (NPA) were taken to study RSV antigens by indirect immunofluorescence.
All hospitalized children had at least two consecutive positive NPA for RSV, and all control children included in the study were negative for RSV in NPA and in the PBMC. Because of the small amount of blood that could be obtained, not all tests were performed in every child. The number of mononuclear cells required for cell phenotyping was 3 × 106, and 6 × 106 for interleukin quantification. Therefore, the number of tests performed in every child depended on the number of mononuclear cells obtained. Informed consent for the study was given by the parents of control and RSV-infected infants. The study was approved by the hospital and Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) ethical committees.
Collection of Blood
Heparinized blood (3 to 5 ml) was collected under sterile conditions from every infant to analyze total leukocytes, differential cells counts, monocytes, and lymphocyte subsets. These cells were cultured to determine cytokine production.
Phenotyping of Blood Mononuclear Cells
Mononuclear cells were separated by a density gradient (Lymphoprep; Nycomed) and counted in a Neubauer chamber, and viability was assessed by Trypan blue dye exclusion. Using a panel of monoclonal reagents to CD45/CD14, CD3/CD19, CD4/CD8, CD4/HLADR, CD4/CD25, CD8/HLADR, CD8/CD25, CD19/HLADR, CD19/CD25, CD14/HLADR, and CD14/CD25, phenotyping was performed. The samples were analyzed on the FACScan flow cytometer (Becton-Dickinson) within 24 h using Lysis software. From each sample 20,000 cells were acquired, and the nonspecific staining, assessed by an isotype control, was adjusted to less than 1%. Mononuclear subsets were expressed as a percentage, and absolute numbers were calculated on the basis of laboratory differential and white blood cell counts.
Detection of RSV Antigens in Blood Mononuclear Cells
Intracellular expression of RSV antigens in blood mononuclear cells
was detected by the indirect immunofluorescence test (IFAT). Briefly, mononuclear cells were permeabilized with 70% ethanol at
20° C, incubated at least 15 min in the cold, and washed twice in
PBS. Infected and noninfected cells were incubated with anti-RSV antibodies for 1 h at 4° C, washed twice in PBS 2% FCS, and incubated with goat antimouse IgG-FITC for 1 h at 4° C. The cells were washed twice in PBS 2% FCS, centrifuged, and resuspended at a concentration of 1 × 106/ml in filtered PBS 2% FCS. A minimum of 20,000 cells
were analyzed on FACScan flow cytometer using Lysys software.
Nonspecific staining was assessed by incubating the cells with the second antibody (16). Samples were also analyzed in a fluorescence microscope (Labophot 2; Nikon, Tokyo, Japan).
Detection of RSV Antigens in NPA
RSV antigens in NPA were detected as previously described (1, 17). Briefly, specimens were obtained by gentle washing of nasopharyngheal secretions and placed on ice while transported to the laboratory. Samples were smeared onto a microscope slide, let dry, and fixed with acetone. RSV antigens were detected by FAT as described before and analyzed in a fluorescence microscope (Labophot 2; Nikon).
Culture of Cells
PBMC from RSV infected and control infants were cultured in flat-bottomed 24-well culture plates (Nunc, Roskilde, Denmark) at a concentration of 1 × 106 cells per well in 600 µl of AIM-V medium, in the
presence or absence of PHA (10 µg/ml) or PWM (1.6 µg/ml) for 24 and 48 h at 37° C with 5% CO2. After culture, cells were centrifuged
and viability was determined by Trypan blue dye exclusion. The supernatant was kept at 20° C prior to IL-4 and IFN- assay.
IFN- and IL-4 Assay
The concentrations of these cytokines were determined in neat mononuclear cell supernatants using an ELISA kit from Endogen (Woburn, MA). The limit of detectability of these assays was about 2 pg/ml. Samples that registered above the standard curve were diluted and reanalyzed. When values were not detectable, the minimum detectable level was used.
Statistical Analysis
The results of cell receptors when expressed as percentages were converted to arcsin or expressed as absolute values. Data were expressed as mean ± SEM and analyzed by Student's upaired t test. Interleukin concentrations were also analyzed by ANOVA and the Neuman-Keuls test. A p < 0.05 was considered to be significant.
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Mononuclear Cell Subpopulations
The mononuclear cell subpopulation was identified with double marker monclonal antibodies to the following cell antigens: CD45/CD14, CD3/CD19, and CD4/CD8. The results are shown in Table 1, expressed as mean ± SEM of cells/mm3 of 12 infected and 12 control infants. In infected compared with control infants there was a significant increase in the proportion and number of CD19+ cells (30.8 versus 15.0%, p < 0.02). In addition, a significant decrease of the CD8+ T-cell subpopulation was observed in infected infants (16.6 versus 22.5%, p < 0.05).
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Mononuclear Cells Bearing Activation Markers
The number of mononuclear cells expressing the CD25 and HLADR activation markers was also determined in seven infected and seven control infants, and the results, expressed as mean ± SEM of cells/mm3, are shown in Table 2. Only the CD8+/CD25+ cell subpopulation was significantly decreased in infected infants compared with control infants, with values of 11 ± 3 versus 27 ± 4, respectively (p < 0.007). No difference was found in the others subpopulations bearing activation markers.
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Production of IL-4
The levels (pg/ml) of IL-4 of cultured PBMC from RSV- infected and control infants in the presence of PHA obtained after 24 and 48 h are shown in Figure 1 (individual values and mean ± SEM).
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IL-4 production by PHA-stimulated PBMC from 12 RSV-infected infants was 0.8 ± 0.1 and 1.1 ± 0.1 at 24 and 48 h, respectively, significantly less (p < 0.01) than from control infants of 1.7 ± 0.3 at 24 h (n = 12) and 2.5 ± 0.4 at 48 h (n = 17).
Production of IFN-
The concentrations (pg/ml) of IFN- in the supernatant of cultured PBMC from RSV-infected and control infants in the
presence of PHA obtained after 24 and 48 h of culturing are
shown in Figure 2 (individual values and mean ± SEM).
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The supernatant of PHA-stimulated cells from (n = 14)
RSV-infected infants had no detectable amount of IFN- at
24 h of culture, and only two of these patients had detectable
levels of IFN- at 48 h. In contrast, IFN- was detectable in
supernatants of PHA-stimulated cells from control infants at a
level of 89 ± 38 and 173 ± 42 at 24 h and 48 h, respectively
(p < 0.02), and they were significantly higher (p < 0.02) than
supernatants from unstimulated cells (Table 3).
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Ratio of IL-4/IFN- Production by PHA-stimulated
PBMC from RSV-infected and Control Infants
The ratio IL-4/IFN- (individual values and the mean ± SEM) by PHA-stimulated PBMC from control infants and
RSV-infected infants after 24 and 48 h of culture are shown in
Figure 3.
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The ratio of IL-4/IFN- (mean ± SEM) from (n = 12)
RSV-infected infants was 0.39 ± 0.05, significantly higher (p < 0.04) than from (n = 12) control infants of 0.2 ± 0.07 at 24 h.
The ratio of IL-4/IFN- from (n = 12) RSV-infected infants
was not different at 48 h from that at 24 h, with values of 0.39 ± 0.05. However, the ratio for (n = 16) control infants fell to
0.07 ± 0.04 at 48 h. The difference in the ratio at 48 h was
highly significant (p < 0.001) between infected and uninfected
groups.
Production of IL-4 by PBMC and the Ratio of
IL-4/IFN- from RSV-infected Infants With and
Without a Family History of Atopy
When RSV-infected infants were divided according to family
histories of atopy (Figure 4), we found a significantly greater (p < 0.02) release of IL-4 at 48 h from PHA-stimulated cells in infants with family antecedents of atopy. No differences in
IFN- production was found between the groups, and therefore the ratio of IL-4/IFN- was higher (p < 0.04) in RSV-
infected infants with family histories of atopy than in infants
with no family histories of atopy.
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The long-term prognosis of RSV bronchial obstruction and bronchopneumonia in early infancy has been intensively debated. Although there is no evidence to support the hypothesis that RSV infections per se are responsible for the development of asthma, recurrent wheezing episodes, increases in airway resistance, bronchial lability to exercise and metacholine, and other lung function abnormalities have been shown 7 to 10 yr after hospitalization for RSV bronchiolitis (8, 18). Although a causal relationship is not clear, the association between IgE development during RSV infection and subsequent episodes of wheezing has been demonstrated (13). Epidemiologic studies have shown that high levels of IgE at 9 mo are predictors of persistent wheezing at 6 yr of age (19). It may be possible that children with severe RSV disease may be those more prone to have sequelae after infection and have a genetic predisposition to a Th-2 type of immune response to RSV, which induces an IgE response to the virus and may become sensitized to allergens during infection.
In the present study, we have shown that infants infected
naturally with RSV showed a predominant humoral Th-2-type
immune response to the infection, based on: (1) increased
number of B-cells; (2) decreased number of CD8 T-cells and
CD8/CD25 (activated cells); (3) functional depression of Th-1
cells, with absence of IFN-; (4) presence of IL-4; (5) significantly elevated ratio of IL-4/IFN-.
Although several factors, including the dose of antigen, the
type of antigen-presenting cell (APC), and the major histocompatibility complex (MHC) Class II haplotype, influence
the differentiation of naive CD4+ T-cells into specific Th subsets, the best characterized factors affecting the development
of Th subsets are cytokines themselves (20). In this study, we
used the term Th-1 type or Th-2 type of response to refer to
responses dominated by IFN- or IL-4, respectively. Other cytokines may influence the type of immune response, the APC-derived cytokine IL-12 strongly drives the differentiation of
Th-1 cells through its potent induction of IFN- production,
and IL-10 and IL-13 inhibit Th-1 cell proliferation. However,
IL-4 and IFN- are the cytokines that best define the function
of Th-2 and Th-1 cells. Th-2 cytokines encourage antibody production, particularly IgE responses, and Th-1 cells are involved in cell-mediated inflammatory reactions and contribute to the pathogenesis of organ-specific autoimmune diseases
(21).
Presently, the most reliable test to identify subjects at high
risk of developing allergy, is the detection of high levels of IgE
antibodies (22). However, recent data suggest that an alteration in the balance of Th-1/Th-2 immune responsiveness occurs in subjects at risk of developing asthma that precedes allergic sensitization, and it occurs during the first year of life.
Reduced production in IFN- by cord-blood cells from infants
with a family history of atopy has been demonstrated (23).
Further, decreased IFN- and IL-2 production by stimulated
PBMC was found during the first year of life in children who
will later become sensitized to Alternaria (24). An elevated serum level of IL-4 in infants who later in life develop atopic disease has also been reported (25). In our study, we found significantly more IL-4 production by PHA-stimulated PBMC in
infants with a family history of atopy, suggesting an increased
capacity for IL-4 production that may be genetic.
The infants selected in this study had a severe disease with bronchial obstruction with or without radiologic findings of bronchopneumonia, suggesting that the severity of the disease could be related to the Th-2-type pattern of response. In animal experiments, Th-2 responses are immunopathogenic, induced mainly by intense pulmonary eosinophilia (26). In infants with RSV infection, eosinophil cationic protein (ECP) in respiratory secretions was associated with the development of wheezing (27).
Many studies have shown that RSV does not induce IFNs
either in vivo or in vitro in cultured cells (6, 28). IFN-
plays a crucial role in inhibiting IgE production induced by
IL-4-stimulated mononuclear cells (31). IL-4 is the most potent interleukin in inducing in B-cells germ-line-
expression
(32). In in vitro culture cells and in experimental animal studies it was demonstrated that IL-4 is critical in the development
of the differentiation of Th-0 to Th-2 response. Natural immune response to some viruses, with high concentrations of
IFN- in absence of IL-4, provides optimal conditions for the
development of Th-1 cells. However, in our study, RSV produced the opposite cytokine profile, with the presence of IL-4
in the absence of IFN-. The presence in the microenvironment of IL-4 in conjunction with undetectable or low concentrations of IFN- seems to represent a favorable condition for
the development of Th-2 cells (33). Our results suggest that,
during severe RSV infections, there is a functional depression
of CD4T cells that is more severe for Th-1 than for Th-2 subpopulations. Consequently, the presence of IL-4 and reduced IFN- would provide a favorable microenvironment for an
IgE response to inhaled or ingested allergens.
It was interesting to find that IL-4 was significantly increased in PWM-stimulated PBMC in RSV-infected infants.
The source of IL-4 is inknown, but it could be B-cells, CD8T,
or CD4 Th-2 cells. PWM is a T-cell-dependent B-cell activator
that has previously been shown to induce IL-4 and IFN- by
PBMC from normal donors (34). In our study, PWM- and
PHA-stimulated PBMC from control infants induced similar
levels of IL-4 (at 24 h) and IFN- (at 48 h). PWM-stimulated
cells from RSV-infected infants induced small amounts of IFN-,
but a significantly increased level of IL-4, whereas PHA-stimulated cells did not release IFN- and maintained the level of
IL-4. The source of the IFN- may be NK or CD8T cells. PHA
preferentially stimulates CD4 T-cells. The imbalance in the cytokine production was found with in vitro mitogen-activated PBMC; RSV-stimulated cells may induce a different response.
Finally, we now have a prospective study to determine how
long after RSV infection the functional depression of CD4
T-cells may persist. In addition, we will determine the duration of the imbalance in the IL-4/IFN- ratio, during which
time allergic sensitization might more easily occur.
In summary, we have found that in infants infected naturally with RSV, and with severe ALRI, there is a functional depression of CD4 T-cells, that is more severe for the Th-1 subpopulation than for the Th-2 subpopulation. This effect would favor a Th-2-type humoral response in these infants, and it could explain the inflammatory response and the lack of T-cell development, with limited protection for subsequent infections, and the Ig-E-induced response, both nonspecific and RSV-specific.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Patricia V. Díaz, Depto. Med. Exp. Oriente, Fac. De Medicina, Universidad de Chile, Avda. Salvador 486, Casilla 16028, Santiago, Chile.
(Received in original form November 13, 1996 and in revised form March 5, 1997).
Acknowledgments: The writers are thankful for the collaboration of nurses and doctors in charge of patients in this study.
Supported by Proyecto Fondecyt 92/0932.
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References |
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REFERENCES
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