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Induction of cytokine mRNA in peripheral blood mononuclear cells of infants after the first dose of measles vaccine
Vaccine 19 (2001) 4896– 4900 www.elsevier.com/locate/vaccine
Induction of cytokine mRNA in peripheral blood mononuclear cells of infants after the first dose of measles vaccine H. Li a,*, C.J. Hickman a, R.F. Helfand a, H. Keyserling b, L.J. Anderson a, W.J. Bellini a a
Respiratory and Enteric Viruses Branch, Measles Section MS. D11, Centers for Disease Control and Pre6ention, 1600 Clifton Road, Atlanta, GA 30333, USA b Department of Pediatrics, Emory Uni6ersity School of Medicine, Atlanta, GA 30333, USA Received 29 July 2000; received in revised form 28 March 2001; accepted 29 March 2001
Abstract To better characterize the cytokine response to measles virus vaccine, we examined the levels of IL-2, IL-4, IL-5, IL-10, IL-12 and g-interferon (g-IFN) in measles virus-stimulated peripheral blood mononuclear cells from 18 donors before and 2 weeks after vaccination. Donors were grouped as seropositive or seronegative on the basis of measles-specific IgM antibody present at 2 weeks postvaccination. After vaccination, similar levels of upregulation of IL-2 and g-IFN mRNA were observed in the two groups. The majority of donors in both groups did not exhibit an increase in measles specific IL-4 or IL-10 mRNA after vaccination. IL-12 mRNA was not induced by measles virus in any of the donors. A statistically significant upregulation of IL-5 mRNA was observed among seropositive (9/13) compared with seronegative (1/5) donors after vaccination (P = 0.09, one tailed Fisher’s test). The observed measles specific induction of IL-5 mRNA is suggestive of a possible association between IL-5 production and an antibody response to measles virus. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cytokine; Infants; Measles
1. Introduction Measles virus (MV), a member of the Morbilli6irus genus of the Paramyxo6iridae family, is the etiologic agent of acute measles, which causes almost one million deaths of infants and children worldwide each year. Vaccination programs have resulted in a major reduction of measles cases in the US; however, the occurrence of measles in individuals with documented seroconversion indicates that the current vaccine occasionally does not provide lifelong protection [1 – 3]. Both cellular and humoral immune mechanisms are involved in protection against measles. Levels of neutralizing antibodies to measles correlate well with protection from measles infection, and maternal antibodies protect infants from infection for several months after * Corresponding author. Tel.: + 1-404-639-4554; fax: +1-404-6394211. E-mail address: [email protected] (H. Li).
birth [1,4]. Children with congenital agammaglobulinemia respond to measles with the usual sequence of symptoms and subsequently develop immunity to reinfection [5,6]. On the other hand, children with specific defects in T cell function often develop severe measles with fatal bacterial or viral complications [7,8]. These observations support a critical role for T cells in both recovery from primary measles infection and protection from subsequent disease. Cytokines are intracellular signaling molecules. These molecules, produced predominately by T cells and macrophages, play an important role in the regulation of immunity and inflammation and coordinate diverse cellular responses, including proliferation and differentiation of hematopoietic cells [9]. CD4+ helper T (Th) cells can be classified as Th1 or Th2 cells on the basis of cytokine secretion patterns [9]. These Th cell populations are functionally distinct and cross-regulatory. Type 1 cytokines include IFN-g, IL-2, and TNF-b and bias a Th cell-dependent immune response toward a
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 2 2 7 - 4
H. Li et al. / Vaccine 19 (2001) 4896–4900
cellular response. Type 2 cytokines include IL-4, IL-5, IL-6, IL-10, and IL-13 and favor the development of a strong humoral immune response. Cytokine alterations have been noted after human infection with viruses, bacteria, and parasites [10– 13]. The cytokine response to an invading microorganism can influence the development of a protective or pathological immune response in the host. Acute measles virus infection induces a predominant Th2-type immune response characterized by spontaneous release of IL-4 [14,15], production of high levels of antibodies to measles virus [16] and temporary suppression of cell-mediated immunity. Immune alterations associated with measles virus infection include decreased in vivo delayed type hypersensitivity (DTH) responses; suppression of in vitro lymphoproliferative responses to MV, mitogen, and recall antigen; increased plasma IgE; and elevated plasma levels of IFN, soluble IL-2 and soluble CD8 [17– 22]. In addition, LPS-stimulated human monocyte cultures infected with live wild-type measles virus exhibited decreased production of IL-12, suggesting a link between cytokine production and measles-associated immune suppression [23]. Currently, most infants in the US are exposed to measles virus through vaccination, which usually results in long-term production of measles-specific antibodies. Some infants, however, fail to mount an immune response after the first dose and less commonly after the second dose, of measles vaccine. Since, Th-cell-produced cytokines are required for B cell differentiation and Ig isotype switching to produce measles-specific antibodies, it is important to understand the cytokine response to measles after the first dose of measles, mumps, and rubella (MMR) combined vaccine. The correspondence between cytokine response and antibody production to measles vaccine has not been investigated earlier. In this study, we examined the levels of measles-specific antibodies and the measles-specific induction of cytokine mRNAs in peripheral blood mononuclear cells (PBMCs) from infants before and after vaccination with the first dose of MMR vaccine.
2. Materials and methods
2.1. Subjects and preparation of PBMCs Thirty infants aged 12– 15 months were inoculated intramuscularly with a single dose of MMR. Five milliliters of peripheral blood was collected from each infant before vaccination and at 2 weeks after vaccination. Subjects were classified as seropositive and seronegative on the basis of antibody production to measles nucleoprotein as determined by enzyme-linked
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immunosorbent assay (ELISA) [24]. PBMCs were isolated from whole blood by using a CPT cell preparation tube (Beckon Dickinson Vacutainer System, Franklin Lakes, NJ) containing sodium citrate gel and density media. After separation, PBMC were washed twice with RPMI (GIBCO Laboratories, Grand Island, NY) supplemented with 2 mM glutamine, 20 mM N-2-hydroxyethylpiperazine-N%-2ethanesulfonic acid (HEPES), 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 10% fetal calf serum (FCS) [25]. Cells were then washed with RPMI without FCS and stimulated with measles virus (Moraten) at a multiplicity of infection (MOI) of 1. Controls consisted of unstimulated PBMCs, mock (uninfected vero cell lysate)-stimulated PBMCs, and PHA (1 mg/ml)-stimulated PBMCs in RPMI without serum. Cells were stimulated at 37 °C for 1 h and then supplemented with RPMI containing human type AB sera such that the final concentration was 5%. Cultures were then incubated at 37 °C for an additional 23 h.
2.2. Virus preparation Measles virus (Moraten) was plaque purified and propagated in Vero cells maintained in 5% FCS-minimum essential medium (MEM) supplemented with 2 mM glutamine, 20 mM HEPES, 100 U of penicillin per ml, and 100 mg of streptomycin per ml. The virus stock was inoculated onto the monolayer of Vero cells in 150-cm2 flasks at an MOI of 0.001. The virus stock was harvested, purified, and titrated as earlier described [26]. Mock antigen was prepared using uninfected Vero cells as described for preparation of measles virus.
2.3. RNA extraction and re6erse transcription RNA was extracted from PBMC pellets by using RNAzol B (Biotex Laboratories, Houston) according to the manufacturer’s instructions. One microgram of extracted RNA was used for reverse transcription. A total of 19 ml of RNA solution was mixed with 4 ml of 12–18 mer poly-dT, (Pharmacia Biotechnology, Piscatway, NJ). The mixture was heated at 65 °C for 5 min. and then cooled on ice for 3 min. The reverse transcription reaction was then carried out by adding 4 ml of 0.1 M dithiothreitol, 20 U of Rnase inhibitor, 4 ml of 10 mM dNTPs, and 8 ml of 5 × reverse transcription (RT) buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2; GIBCO BRL, Life Technologies, Inc., Gaithersburg, MD). The RT mixture was incubated at 37 °C for 2 h. The reaction was stopped by heating at 70 °C for 10 min. Fifty microliters of the cDNA was diluted with 50 ml of distilled H2O and then stored at − 20 °C.
H. Li et al. / Vaccine 19 (2001) 4896–4900
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2.4. PCR and quantitation of PCR products
measurable amounts of antibody to measles virus at 2 weeks after vaccination. PBMCs obtained from 13 donors in the IgM-positive group and all five infants in the IgM-negative group were stimulated with measles virus or control antigens and then analyzed for cytokine mRNA induction by PCR. Approximately, 40% of seronegative individuals and 60% of seropositive individuals had a measles-specific increase in IL-2 or IFN-g (Table 1). Twenty-three percent (3/13) of the seropositive infants and none of the seronegative infants (0/5) exhibited an IL-4 LUM index value of \ 2 after vaccination (P= 0.35, Fisher’s one tailed test). IL-12 mRNA was not specifically induced by measles virus in any of the infants (data not shown). In contrast, IL-5 mRNA was induced by measles virus in 70% (9/13) of the seropositive infants, but only 20% (1/5) of the seronegative infants (Fig. 1) (P= 0.09, Fisher’s one tailed test). The LUM value for measles virus-induced IL-5 mRNA in the seronegative group was significantly lower than that for the seropositive group (PB0.001 student’s t-test). The LUM value for IL-5 mRNA induced by PHA in the seronegative infants was similar to that for the seropositive infants (P\ 0.25). The three seropositive individuals who were negative for measles virus-induced IL-5 mRNA had the highest levels of measles virus-induced IL-2 mRNA expression (Fig. 2). The seronegative persons who were negative for measles virus-induced IL-5 mRNA did not have high levels of measles virus-induced IL-2 mRNA.
The PCR was set up for a 50-ml reaction volume and consisted of 5 ml of 10 × PCR buffer (100 mM Tris– HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), 200 mM dNTPs, 10 pmol each of forward and reverse primers, and 2.5 U of Taq polymerase (Perkin– Elmer). The reaction mixture was denatured for 75 s at 94 °C and then annealed for 75 s at the appropriate annealing temperature. The annealing temperature was 55 °C for IFN-g and IL-2, 65 °C for IL-4, IL-5, IL-10, and IL-12 [11], and 60 °C for the positive control porphobilinogen deaminase (PBGD) [27]. Cytokine cDNA was amplified for 35 cycles, and PBGD cDNA was amplified for 30 cycles. cDNA used for PCR was equilibrated according to the efficiency of amplification of PBGD cDNA, a control template. Five microliters of PCR product was hybridized with 10 pmol of ruthenium-labeled probe complementary to an internal sequence of the amplified strand of cytokine cDNA that was extended from the biotin-labeled primer. The strand with the biotin labeled primer was captured by streptavidin-coated magnetic beads (Dynal, Great Neck, NY), and the amount of hybridized probe was quantitated by measurement of the electrochemiluminesence generated from ruthenium chelate, using a Perkin –Elmer QPCR system 5000 analyzer [11]. The luminescence index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value. A luminescence index value of two was used as a cutoff for quantitation of PCR products [28]. The value of two was calculated by comparing the distributions of measles-induced cytokine levels (measured as a luminescence index) in prevaccinated donors and postvaccinated donors. The majority of prevaccinated donors had an index that was under 1.5. An index greater than two was defined as positive. Levels of measles specific IgM to the nucleoprotein of measles virus was measured pre and postvaccination by a capture-antibody ELISA that has been described earlier [24].
4. Discussion To date, most studies measuring cytokine production in response to measles virus infection or vaccination have detected these cytokines in plasma or from cultures of PBMCs manipulated by the addition of cytokine or mitogen [10,14,20]. Alterations in mitogen responses [7,18], suppression of DTH reaction [22], and altered cytokine production [14,20,21] have been reported in natural measles virus infection and measlesvaccinated individuals. Induction of IFN-g, IL-2, IL-4 and IL-10 mRNA was not significantly different between the measles seropositive and seronegative infants. However, a correspondence between antibody production and IL-5 mRNA upregulation after measles virus stimulation was observed in this study. IL-5 mRNA
3. Results Twenty-five of 30 infants developed measles specific IgM antibodies. Five of 30 infants did not produce
Table 1 Specific cytokine mRNA induction by measles virus among seropositive and seronegative infants after vaccination Group
IFN-g
IL-2
IL-4
IL-5
IL-10
IL-12
Seropositive Seronegative
61a 40
61 40
23 0
69.2 20
15.4 20
0 0
a
Percentage of cytokine positive individuals in both seropositive and seronegative infant groups.
H. Li et al. / Vaccine 19 (2001) 4896–4900
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Fig. 1. Induction of IL-5 mRNA in PBMC from measles-vaccinated infants. PBMC from measles vaccinated infants were stimulated with mock control, measles virus (moraten, MOI = 1) or PHA (1 mg/ml) for 24 h. Induction of IL-5 mRNA (expressed as a LUM index) following measles virus stimulation is shown for seropositive infants (A) and seronegative infants (B). Positive control induction of IL-5 mRNA by PHA is shown for seropositive infants (C) and seronegative infants (D). The LUM index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value.
was induced from 70% of seropositive infants but from only 20% of the seronegative infants indicating that measles virus-specific IL-5 mRNA induction maybe related to specific antibody production after measles vaccination. IL-5 is a product of activated T lymphocytes and demonstrates activity on eosinophils, basophils, B cells, and thymocytes [29]. It stimulates IgA and IgM secretion from B cells and acts as a chemotactic and activating factor for eosinophils [30]. Interestingly, the PBMC from antibody-positive persons who failed to produce IL-5 after measles virus stimulation had the highest levels of IL-2 induced by measles virus. This finding might be explained by the reciprocal regulation of Th1 and Th2 cells [9]. It is possible that infants with low levels of IL-5 but high levels of IL-2 may have strong cellular (proliferative and CTL) responses to measles virus antigens. Studies are continuing in our laboratory to investigate the relationship between antibody response and cellular immune response in seronegative and seropositive vaccines. IL-12 mRNA was not induced by measles virus in either seropositive or seronegative groups. The lack of IL-12 production might help to explain the temporary suppression of cellular immunity associated with both natural measles infection and vaccination. One limitation of the study is that the data represent only one point in time. It is possible that serial specimens that define the kinetics of measles virus-induced
cytokine production might provide a clearer link between antibody response and cytokines in PBMCs. The results of this study do, however, suggest a direction and methodology for future studies that should help clarify knowledge of the immune response to measles vaccines and provide clues to the basis for measles virus-induced immunosuppression.
Fig. 2. Induction of IL-2 and IL-5 mRNA by measles virus in seropositive infants. PBMC from measles vaccinated infants were stimulated with mock control, measles virus (moraten, MOI =1) or PHA (1 mg/ml) for 24 h. Levels of measles virus-specific IL-2 mRNA (open bars) and IL-5 mRNA (striped bars) expression are shown for measles seropositive infants. Values are expressed as a LUM index. The LUM index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value.
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References [1] Black FL. Measles active passive immunity in a worldwide perspective. Prog Med Virol 1989;36:1 –33. [2] Mathias RG, Meekison WG, Arcand TA, Schechter MT. The role of secondary vaccine failure in measles outbreaks. Am J Public Health 1989;79:475 –8. [3] Markowitz LE, Preblud SR, Fine PEM, Orenstein WA. Duration of live measles vaccine-induced immunity. Pediatr Infect Dis J 1990;9:101 – 10. [4] Coovadia HM, Wesley A, Brain P. Immunologic events in acute measles influencing outcome. Arch Dis Child 1978;53:861 – 7. [5] Good RA, Zak SJD. Disturbances in gamma globulin synthesis as ‘experiments of nature’. Pediatrics 1956;18:109 –49. [6] Burnet FM. Measles as an index of immunological function. Lancet 1968;2:610 – 3. [7] Krasinski K, Borkowsky W. Measles and measles immunity in child infected with human immunodeficiency virus. Am J D 1989;261:2512 – 6. [8] Monsfo WJ, Haslam DB, Roberts RL, Zaki SR, Bellini WJ, Coffin CM. Disseminated measles infection following vaccination in severe combined immune deficiency syndrome. J Pediatr 1994;124:273 – 6. [9] Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996;17:138 –46. [10] Schneider-Schaulies J, Schneider-Schaulies S, ter Meulen V. Differential induction of cytokines by primary and persistent measles virus infections in human glial cells. Virolology 1993;195:219 – 28. [11] Anderson LJ, Tsou C, Potter C, Keyserling HH, Smith TF, Ananaba G, Bangham CR. Cytokine response to respiratory syncytial virus stimulation of human peripheral blood mononuclear cells. J Infect Dis 1994;170:1201 – 8. [12] Yamamura M, Uyemura K, Dean RJ, Weinberg K, Rea TH, Bloom BR, Modlin RL. Defining protective responses to pathogen: Cytokine profiles in leprosy lesions. Science (5029) 277 – 79. [13] Bogdan C, Gessner A, Rollinghoff M. Cytokines in Leishmaniasis: a complex network of stimulatory and inhibitory interactions. Immunobiology 1993;189:356 – 96. [14] Ward BJ, Griffin DE. Changes in cytokine production after measles virus vaccination: predominant production of IL-4 suggests induction of a Th2 response. Clin Immunol Immunopathol 1993;67:171 – 7. [15] Griffin DE, Ward BJ. Differential CD4 T cell activation in measles. J Infect Dis 1993;168:275 – 81. [16] Norrby E, Gollmar Y. Appearance and persistence of antibodies against different virus components after regular measles infections. Infect Immun 1972;6:240 –7. [17] Ward BJ, Johnson RT, Vaisberg A, Jauregui E, Griffin DE. Cytokine production in vitro and the lymphoproliferative defect of natural measles virus infection. Clin Immunol Immunopathol 1991;61:236 – 48.
[18] Hirsch RL, Mokhtarin F, Griffin DE, Brooks B, Hess J, Johnson RT. Measles virus vaccination of measles seropositive individuals suppresses lymphocyte proliferation and chemotactic factor production. Clin Immunol Immunopathol 1981;21:341 – 50. [19] Ward BJ, Johnson RT, Vaisberg A, Jauregui E, Griffin DE. Spontaneous proliferation of peripheral mononuclear cells in natural measles virus infection: identification of dividing cells and correlation with mitogen responsiveness. Clin Immunol Immunopathol 1990;55:315 – 26. [20] Griffin DE, Ward BJ, Jauregui E, Johnson RT, Vaisberg A. Immune activation during measles: interferon-g and neopterin in plasma and cerebrospinal fluid in complicated and uncomplicated disease. J Infect Dis 1990;161:449 – 53. [21] Leopardi R, Vainionpaa R, Hurme M, Siljander P, Salmi AA. Measles virus infection enhances IL-g but reduces tumor necrosis factor-g expression in human monocytes. J Immunol 1992;149:2397 – 401. [22] Brody JA, McAlister R. Depression of tuberculin sensitivity following measles vaccination. Am Rev Respir Dis 1964;90:607 – 11. [23] Karp CL, Wysocka M, Wahl LM, Ahearn JM, Cuomo PJ, Sherry B, Trinchieri G, Griffin DE. Mechanism of suppression of cell-mediated immunity by measles virus. Science 1996;273:228 – 31. [24] Erdman DD, Anderson LJ, Adams DR, Steward JA, Markowitz LE, Bellini WJ. Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus. J Clin Microbiol 1991;29:1466 – 71. [25] Hickman CJ, Khan AS, Rota PA, Bellini WJ. Use of synthetic peptides to identify measles nucleoprotein T – cell epitopes in vaccinated and naturally infected humans. Virology 1997;235:386 – 97. [26] Bellini WJ, McFarlin DE, Silver GD, Mingioli ES, McFarland HF. Immune reactivity of the purified hemagglutinin of measles. Infect Immun 1981;32:1051 – 7. [27] Finke J, Fritzen R, Ternes P, Lange N, Dolken G. An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, parafin-embedded tissues by PCR. Biotechniques 1993;14:448 – 53. [28] Warris M, Tsou C, Erdman DD, Zaki SR, Anderson LJ. Respiratory syncytial virus infection in Balb/C mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant Th2-like cytokine pattern. J Virol 1996;70:2852 – 60. [29] Schrezenmeier H, Thome SD, Tewald F, Fleischer B, Raghavachar A. Interleukin-5 is the predominant eosinophilopoietin produced by cloned T lymphocytes in hypereosinophilic syndrome. Exp Hematol 1993;21:358 – 65. [30] Rasmussen R, Takatsu K, Harada N, Takahashi T, Bottomly K. T cell-dependent hapten-specific and polyclonal B cell responses require release of interleukin- 5. J Immunol 1988;140:705 –12.
Induction of cytokine mRNA in peripheral blood mononuclear cells of infants after the first dose of measles vaccine H. Li a,*, C.J. Hickman a, R.F. Helfand a, H. Keyserling b, L.J. Anderson a, W.J. Bellini a a
Respiratory and Enteric Viruses Branch, Measles Section MS. D11, Centers for Disease Control and Pre6ention, 1600 Clifton Road, Atlanta, GA 30333, USA b Department of Pediatrics, Emory Uni6ersity School of Medicine, Atlanta, GA 30333, USA Received 29 July 2000; received in revised form 28 March 2001; accepted 29 March 2001
Abstract To better characterize the cytokine response to measles virus vaccine, we examined the levels of IL-2, IL-4, IL-5, IL-10, IL-12 and g-interferon (g-IFN) in measles virus-stimulated peripheral blood mononuclear cells from 18 donors before and 2 weeks after vaccination. Donors were grouped as seropositive or seronegative on the basis of measles-specific IgM antibody present at 2 weeks postvaccination. After vaccination, similar levels of upregulation of IL-2 and g-IFN mRNA were observed in the two groups. The majority of donors in both groups did not exhibit an increase in measles specific IL-4 or IL-10 mRNA after vaccination. IL-12 mRNA was not induced by measles virus in any of the donors. A statistically significant upregulation of IL-5 mRNA was observed among seropositive (9/13) compared with seronegative (1/5) donors after vaccination (P = 0.09, one tailed Fisher’s test). The observed measles specific induction of IL-5 mRNA is suggestive of a possible association between IL-5 production and an antibody response to measles virus. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cytokine; Infants; Measles
1. Introduction Measles virus (MV), a member of the Morbilli6irus genus of the Paramyxo6iridae family, is the etiologic agent of acute measles, which causes almost one million deaths of infants and children worldwide each year. Vaccination programs have resulted in a major reduction of measles cases in the US; however, the occurrence of measles in individuals with documented seroconversion indicates that the current vaccine occasionally does not provide lifelong protection [1 – 3]. Both cellular and humoral immune mechanisms are involved in protection against measles. Levels of neutralizing antibodies to measles correlate well with protection from measles infection, and maternal antibodies protect infants from infection for several months after * Corresponding author. Tel.: + 1-404-639-4554; fax: +1-404-6394211. E-mail address: [email protected] (H. Li).
birth [1,4]. Children with congenital agammaglobulinemia respond to measles with the usual sequence of symptoms and subsequently develop immunity to reinfection [5,6]. On the other hand, children with specific defects in T cell function often develop severe measles with fatal bacterial or viral complications [7,8]. These observations support a critical role for T cells in both recovery from primary measles infection and protection from subsequent disease. Cytokines are intracellular signaling molecules. These molecules, produced predominately by T cells and macrophages, play an important role in the regulation of immunity and inflammation and coordinate diverse cellular responses, including proliferation and differentiation of hematopoietic cells [9]. CD4+ helper T (Th) cells can be classified as Th1 or Th2 cells on the basis of cytokine secretion patterns [9]. These Th cell populations are functionally distinct and cross-regulatory. Type 1 cytokines include IFN-g, IL-2, and TNF-b and bias a Th cell-dependent immune response toward a
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 2 2 7 - 4
H. Li et al. / Vaccine 19 (2001) 4896–4900
cellular response. Type 2 cytokines include IL-4, IL-5, IL-6, IL-10, and IL-13 and favor the development of a strong humoral immune response. Cytokine alterations have been noted after human infection with viruses, bacteria, and parasites [10– 13]. The cytokine response to an invading microorganism can influence the development of a protective or pathological immune response in the host. Acute measles virus infection induces a predominant Th2-type immune response characterized by spontaneous release of IL-4 [14,15], production of high levels of antibodies to measles virus [16] and temporary suppression of cell-mediated immunity. Immune alterations associated with measles virus infection include decreased in vivo delayed type hypersensitivity (DTH) responses; suppression of in vitro lymphoproliferative responses to MV, mitogen, and recall antigen; increased plasma IgE; and elevated plasma levels of IFN, soluble IL-2 and soluble CD8 [17– 22]. In addition, LPS-stimulated human monocyte cultures infected with live wild-type measles virus exhibited decreased production of IL-12, suggesting a link between cytokine production and measles-associated immune suppression [23]. Currently, most infants in the US are exposed to measles virus through vaccination, which usually results in long-term production of measles-specific antibodies. Some infants, however, fail to mount an immune response after the first dose and less commonly after the second dose, of measles vaccine. Since, Th-cell-produced cytokines are required for B cell differentiation and Ig isotype switching to produce measles-specific antibodies, it is important to understand the cytokine response to measles after the first dose of measles, mumps, and rubella (MMR) combined vaccine. The correspondence between cytokine response and antibody production to measles vaccine has not been investigated earlier. In this study, we examined the levels of measles-specific antibodies and the measles-specific induction of cytokine mRNAs in peripheral blood mononuclear cells (PBMCs) from infants before and after vaccination with the first dose of MMR vaccine.
2. Materials and methods
2.1. Subjects and preparation of PBMCs Thirty infants aged 12– 15 months were inoculated intramuscularly with a single dose of MMR. Five milliliters of peripheral blood was collected from each infant before vaccination and at 2 weeks after vaccination. Subjects were classified as seropositive and seronegative on the basis of antibody production to measles nucleoprotein as determined by enzyme-linked
4897
immunosorbent assay (ELISA) [24]. PBMCs were isolated from whole blood by using a CPT cell preparation tube (Beckon Dickinson Vacutainer System, Franklin Lakes, NJ) containing sodium citrate gel and density media. After separation, PBMC were washed twice with RPMI (GIBCO Laboratories, Grand Island, NY) supplemented with 2 mM glutamine, 20 mM N-2-hydroxyethylpiperazine-N%-2ethanesulfonic acid (HEPES), 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 10% fetal calf serum (FCS) [25]. Cells were then washed with RPMI without FCS and stimulated with measles virus (Moraten) at a multiplicity of infection (MOI) of 1. Controls consisted of unstimulated PBMCs, mock (uninfected vero cell lysate)-stimulated PBMCs, and PHA (1 mg/ml)-stimulated PBMCs in RPMI without serum. Cells were stimulated at 37 °C for 1 h and then supplemented with RPMI containing human type AB sera such that the final concentration was 5%. Cultures were then incubated at 37 °C for an additional 23 h.
2.2. Virus preparation Measles virus (Moraten) was plaque purified and propagated in Vero cells maintained in 5% FCS-minimum essential medium (MEM) supplemented with 2 mM glutamine, 20 mM HEPES, 100 U of penicillin per ml, and 100 mg of streptomycin per ml. The virus stock was inoculated onto the monolayer of Vero cells in 150-cm2 flasks at an MOI of 0.001. The virus stock was harvested, purified, and titrated as earlier described [26]. Mock antigen was prepared using uninfected Vero cells as described for preparation of measles virus.
2.3. RNA extraction and re6erse transcription RNA was extracted from PBMC pellets by using RNAzol B (Biotex Laboratories, Houston) according to the manufacturer’s instructions. One microgram of extracted RNA was used for reverse transcription. A total of 19 ml of RNA solution was mixed with 4 ml of 12–18 mer poly-dT, (Pharmacia Biotechnology, Piscatway, NJ). The mixture was heated at 65 °C for 5 min. and then cooled on ice for 3 min. The reverse transcription reaction was then carried out by adding 4 ml of 0.1 M dithiothreitol, 20 U of Rnase inhibitor, 4 ml of 10 mM dNTPs, and 8 ml of 5 × reverse transcription (RT) buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2; GIBCO BRL, Life Technologies, Inc., Gaithersburg, MD). The RT mixture was incubated at 37 °C for 2 h. The reaction was stopped by heating at 70 °C for 10 min. Fifty microliters of the cDNA was diluted with 50 ml of distilled H2O and then stored at − 20 °C.
H. Li et al. / Vaccine 19 (2001) 4896–4900
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2.4. PCR and quantitation of PCR products
measurable amounts of antibody to measles virus at 2 weeks after vaccination. PBMCs obtained from 13 donors in the IgM-positive group and all five infants in the IgM-negative group were stimulated with measles virus or control antigens and then analyzed for cytokine mRNA induction by PCR. Approximately, 40% of seronegative individuals and 60% of seropositive individuals had a measles-specific increase in IL-2 or IFN-g (Table 1). Twenty-three percent (3/13) of the seropositive infants and none of the seronegative infants (0/5) exhibited an IL-4 LUM index value of \ 2 after vaccination (P= 0.35, Fisher’s one tailed test). IL-12 mRNA was not specifically induced by measles virus in any of the infants (data not shown). In contrast, IL-5 mRNA was induced by measles virus in 70% (9/13) of the seropositive infants, but only 20% (1/5) of the seronegative infants (Fig. 1) (P= 0.09, Fisher’s one tailed test). The LUM value for measles virus-induced IL-5 mRNA in the seronegative group was significantly lower than that for the seropositive group (PB0.001 student’s t-test). The LUM value for IL-5 mRNA induced by PHA in the seronegative infants was similar to that for the seropositive infants (P\ 0.25). The three seropositive individuals who were negative for measles virus-induced IL-5 mRNA had the highest levels of measles virus-induced IL-2 mRNA expression (Fig. 2). The seronegative persons who were negative for measles virus-induced IL-5 mRNA did not have high levels of measles virus-induced IL-2 mRNA.
The PCR was set up for a 50-ml reaction volume and consisted of 5 ml of 10 × PCR buffer (100 mM Tris– HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin), 200 mM dNTPs, 10 pmol each of forward and reverse primers, and 2.5 U of Taq polymerase (Perkin– Elmer). The reaction mixture was denatured for 75 s at 94 °C and then annealed for 75 s at the appropriate annealing temperature. The annealing temperature was 55 °C for IFN-g and IL-2, 65 °C for IL-4, IL-5, IL-10, and IL-12 [11], and 60 °C for the positive control porphobilinogen deaminase (PBGD) [27]. Cytokine cDNA was amplified for 35 cycles, and PBGD cDNA was amplified for 30 cycles. cDNA used for PCR was equilibrated according to the efficiency of amplification of PBGD cDNA, a control template. Five microliters of PCR product was hybridized with 10 pmol of ruthenium-labeled probe complementary to an internal sequence of the amplified strand of cytokine cDNA that was extended from the biotin-labeled primer. The strand with the biotin labeled primer was captured by streptavidin-coated magnetic beads (Dynal, Great Neck, NY), and the amount of hybridized probe was quantitated by measurement of the electrochemiluminesence generated from ruthenium chelate, using a Perkin –Elmer QPCR system 5000 analyzer [11]. The luminescence index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value. A luminescence index value of two was used as a cutoff for quantitation of PCR products [28]. The value of two was calculated by comparing the distributions of measles-induced cytokine levels (measured as a luminescence index) in prevaccinated donors and postvaccinated donors. The majority of prevaccinated donors had an index that was under 1.5. An index greater than two was defined as positive. Levels of measles specific IgM to the nucleoprotein of measles virus was measured pre and postvaccination by a capture-antibody ELISA that has been described earlier [24].
4. Discussion To date, most studies measuring cytokine production in response to measles virus infection or vaccination have detected these cytokines in plasma or from cultures of PBMCs manipulated by the addition of cytokine or mitogen [10,14,20]. Alterations in mitogen responses [7,18], suppression of DTH reaction [22], and altered cytokine production [14,20,21] have been reported in natural measles virus infection and measlesvaccinated individuals. Induction of IFN-g, IL-2, IL-4 and IL-10 mRNA was not significantly different between the measles seropositive and seronegative infants. However, a correspondence between antibody production and IL-5 mRNA upregulation after measles virus stimulation was observed in this study. IL-5 mRNA
3. Results Twenty-five of 30 infants developed measles specific IgM antibodies. Five of 30 infants did not produce
Table 1 Specific cytokine mRNA induction by measles virus among seropositive and seronegative infants after vaccination Group
IFN-g
IL-2
IL-4
IL-5
IL-10
IL-12
Seropositive Seronegative
61a 40
61 40
23 0
69.2 20
15.4 20
0 0
a
Percentage of cytokine positive individuals in both seropositive and seronegative infant groups.
H. Li et al. / Vaccine 19 (2001) 4896–4900
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Fig. 1. Induction of IL-5 mRNA in PBMC from measles-vaccinated infants. PBMC from measles vaccinated infants were stimulated with mock control, measles virus (moraten, MOI = 1) or PHA (1 mg/ml) for 24 h. Induction of IL-5 mRNA (expressed as a LUM index) following measles virus stimulation is shown for seropositive infants (A) and seronegative infants (B). Positive control induction of IL-5 mRNA by PHA is shown for seropositive infants (C) and seronegative infants (D). The LUM index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value.
was induced from 70% of seropositive infants but from only 20% of the seronegative infants indicating that measles virus-specific IL-5 mRNA induction maybe related to specific antibody production after measles vaccination. IL-5 is a product of activated T lymphocytes and demonstrates activity on eosinophils, basophils, B cells, and thymocytes [29]. It stimulates IgA and IgM secretion from B cells and acts as a chemotactic and activating factor for eosinophils [30]. Interestingly, the PBMC from antibody-positive persons who failed to produce IL-5 after measles virus stimulation had the highest levels of IL-2 induced by measles virus. This finding might be explained by the reciprocal regulation of Th1 and Th2 cells [9]. It is possible that infants with low levels of IL-5 but high levels of IL-2 may have strong cellular (proliferative and CTL) responses to measles virus antigens. Studies are continuing in our laboratory to investigate the relationship between antibody response and cellular immune response in seronegative and seropositive vaccines. IL-12 mRNA was not induced by measles virus in either seropositive or seronegative groups. The lack of IL-12 production might help to explain the temporary suppression of cellular immunity associated with both natural measles infection and vaccination. One limitation of the study is that the data represent only one point in time. It is possible that serial specimens that define the kinetics of measles virus-induced
cytokine production might provide a clearer link between antibody response and cytokines in PBMCs. The results of this study do, however, suggest a direction and methodology for future studies that should help clarify knowledge of the immune response to measles vaccines and provide clues to the basis for measles virus-induced immunosuppression.
Fig. 2. Induction of IL-2 and IL-5 mRNA by measles virus in seropositive infants. PBMC from measles vaccinated infants were stimulated with mock control, measles virus (moraten, MOI =1) or PHA (1 mg/ml) for 24 h. Levels of measles virus-specific IL-2 mRNA (open bars) and IL-5 mRNA (striped bars) expression are shown for measles seropositive infants. Values are expressed as a LUM index. The LUM index was calculated by dividing the measles induced sample luminosity value by the mock control luminosity value.
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