591
MAJOR ARTICLES
Peripheral Blood Mononuclear Cell Interleukin-2 and Interferon-" Interferon-'Y Production, Cytotoxicity, and Antigen-Stimulated Blastogenesis during Experimental Rhinovirus Infection Judith Hsia, Usia, Allan L. Goldstein, Gary L. Simon, Marcelo Sztein,· and Frederick G. Hayden
Depanments of Biochemistry and Medicine, Medicine. the George From the Departments Uflshington University School of Medicine and Health Sciences, "Ubshington Uflshington. DC, and the Departments Depanments of Internal Medicine and "Ubshington, Pathology, University of Virginia School of Medicine, Medicine. Charlottesville
To determine whether rhinovirus infection induced a systemic cellular immune response in humans, specific antigen-stimulated blastogenesis, natural killer cell activity, and mitogenstimulated production of interleukin-2 and interferon--y by peripheral blood mononuclear cells (pBMC) (PBMC) were quantified during experimental rhinovirus infection of normal volunteers. Phytohemagglutinin-stimulated interleukin-2 production by PBMC collected on day 5 after rhinovirus inoculation was increased fourfold compared with production by PBMC collected before rhinovirus challenge (P < .05); phytohemagglutinin-stimulated interferon--y interferon-)' production was doubled (P < .05). An inverse relationship was observed between the increase in interleukin-2 production and both nasal mucus production (P < .02) and the number of days virus was cultured from nasal washings (P < .02). Natural killer cell-mediated cytotoxicity of PBMC collected on day nasal 5 after rhinovirus challenge was also increased (P < .01) compared with preinfection levels, as was specific antigen-stimulated blastogenesis on day 21 (P < .05). The extent of blastogenic response correlated directly with both mucus production (P < .05) and the number of days virus was cultured from nasal washings (P < .05). These observations are consistent with the hypothesis that rhinovirus infection results in activation of a systemic cellular immune response. Upper respiratory tract infections are the most common rhinoviruse~ are responsible for of human viral illnesses, and rhinoviruses. [1, 2]. Rhinovirus colds are 30 % of these infections [I, at least 30% prejumed pre~umed to be local rather than systemic infections; the immune responses responsible for successful host defense against this agent have not been fully defined. Local and systemic neutralizing antibody is usually not detectable until after resolution of illness, suggesting that alternate host defense mechanisms combat acute infection. Because of acute decreases in peripheral blood T lymphocyte counts during rhinovirus infection, it has been postulated that lymphocyte subsets might migrate to the nasal epithelium to provide in
Received 18 August 1989; revised 2 April 1990. Informed consent was obtained from all study subjects; guidelines of the George Washington University Medical Center Human Research Committee and the University of Virginia Human Investigation Committee were followed. Grant support: National Institutes of Health (CA-24974 to A. L. G., CA42219 to M. B. S., and HL-41507 to 1. H.), Aspirin Foundation of America, and Richardson-Vicks USA. 1. J. H. is a Pfizer Scholar. Reprints and correspondence: Dr. Judith Hsia, Department of Medicine, the George Washington University, 2150 Pennsylvania Ave., NW, WashingWashing, ton, DC 20037. * Present address: Center for Vaccine Development, University of Maryland, Baltimore. The Journal of Infectious DIseases Diseases 1990;162:591-597 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6203-0003$01.00
situ immune protection [3]. Whether these cells would exert antiviral effects through cytokine production, cytotoxic activity, or other means remains to be established. It also has been suggested that immune responses may contribute to symptoms associated with infection, since some of the side effects of treatment with recombinant interferons mimic cold symptoms [4]. To characterize the systemic cellular immune responses during rhinovirus infection and the relationship of those responses to clinical manifestations of infection, we evaluated mitogeninduced production of interleukin-2 and interferon-')' interferon--y by peripheral blood mononuclear cells (PBMC) collected during experimental rhinovirus colds in healthy adult volunteers. Natural killer cell-mediated cytotoxicity and blastogenic response ofPBMCto of PBMC to specific rhinovirus antigens were also assessed, and the relationship between changes in PBMC function and cold symptoms, mucus production, virus shedding, and antibody titers was determined.
Methods Subjects and virus infection. Healthy adult volunteers with titers of serum neutralizing antibody to an unnumbered rhinovirus sub~1:2 were recruited for participation in two clinical type (Hanks) of of~1:2 trials. Individuals with upper respiratory tract illnesses within 2 weeks or who were concurrently taking any medications were excluded. Volunteers were housed individually in motel rooms for 5 days be-
592
Hsia et aI.
ginning on the day of viral challenge (day 1 of the study). An unnumbered rhinovirus subtype (Hanks) was administered by intranasal drops (OJ (0.1 ml/nostril); the total inoculum was "'200 f\J200 50% tissue culture infectious doses (TCIDso) per subject. In the first trial, 20 volunteers were randomly assigned to receive oral aspirin, 325 mg (Miles Laboratories, Washington, DC), or placebo every other day for three doses beginning 24 h before virus challenge. In the second study 20 volunteers were randomized to receive another nonsteroidal antiinflammatory agent or placebo. In this report, only results obtained from the placebo recipients in these trials are included. Surveillance and sampling. Clinical symptoms were monitored during the 5 days after viral challenge, and nasal secretions were weighed by previously described methods [5, 6]. The occurrence of colds was detennined determined by previously described modifications [5, 6] of the method of Jackson et al. [7]. Rates of infection were determined by virus isolation from nasal washings and by measurements of serum neutralizing antibody in paired specimens obtained on the day before viral challenge and 3 weeks later. A fourfold rise in sewas accepted as evirum titer of homotypic neutralizing antibody 'WaS dence of rhinovirus infection. In the first study, sera and heparinized blood samples were collected before and 5 days after virus inoculadetermining serum interferon-')' levels and isolating PBMC tion for detennining on lymphocyte separation medium (Organon Teknika, West Chester, PA) [8]. Heparinized blood samples were also collected 21 days after viral challenge. In the second trial, heparinized blood samples for PBMC isolation were collected before virus inoculation (day 1) and on days 3 and 5. Assays on samples from individual volunteers were run concurrently. Cytokine assays. PBMC were washed, adjusted to 107 cells/ml, (l°C/min) in RPMI 1640 medium (GffiCO, (GmCO, Grand Israte-frozen OOC/min) l1g/ml gentamiland, NY) with 25 mM HEPES buffer, pH 7.5, 10 flglml (HRPMI) supplemented with 60% human cin, and 2 mM glutamine (HRPMl) AB serum·and 20% dimethyl sulfoxide (Fisher Scientific, Columbia, MD) in a linear rate freezer to -70°C, and stored in liquid nitrogen. For immunoassays, frozen PBMC (1 ml) were gently agitated in a water bath (40°C) until just melted. After addition of 1I ml of 20% human serum in HRPMI and incubation (10 min at room temperature), PBMC were transferred to 15-ml conical tubes and incubated with an additional 10 ml of 20% serum (15 min at room temperature). Cells were then washed twice in HRPMI for subsequent cytokine, natural killer cell activity, and blastogenesis assays. determined by trypan blue exclusion, was 85%-90%. Viability, detennined interferon-')' experiments, duplicate samples of PBMC (0.5 For interferon.'Y x 1()6 cells/ml) were incubated in HRPMI with phytohemaggluti#A8!ml, nin (Burroughs Wellcome, Research Triangle Park, NC), 1 pglml, and 1% human AB serum in 96-well flat-bottomed microtiter plates (Nunc, Kamstrup, Denmark) at 37°C in 5% C02: 95% humidified To determine whether cytokine production had been maximally air. Th J.tg/ml (Alpha One Biostimulated, thymosin fraction 5 (TF5), 300 JLg/ml medicals, Washington, DC), a partially purified calf thymic preparation [9], was added to some wells after 48 h. Supernatants were collected at 72 h and stored (-20°C) for interferon-')' interferon-'Y radioimmunoassay (Centocor, Malvern, PA). For interleukin-2 experiments, samples of PBMC (106 cells/ml) J.tg/ml, and were incubated in HRPMI with phytohemagglutinin, 2 JLg/ml, 1% AB serum with or without TF5 in 24-well tissue culture plates 1% (Costar, Cambridge, MA) for 24 h. PBMC were removed by centrifugation (800 g, 12 min at room temperature), and supernatants
lID 1990;162 (September)
interleukin-2-. >-
00
0
CI:l
0
0
..
~ :i'
.E oS
MAJOR ARTICLES
Peripheral Blood Mononuclear Cell Interleukin-2 and Interferon-" Interferon-'Y Production, Cytotoxicity, and Antigen-Stimulated Blastogenesis during Experimental Rhinovirus Infection Judith Hsia, Usia, Allan L. Goldstein, Gary L. Simon, Marcelo Sztein,· and Frederick G. Hayden
Depanments of Biochemistry and Medicine, Medicine. the George From the Departments Uflshington University School of Medicine and Health Sciences, "Ubshington Uflshington. DC, and the Departments Depanments of Internal Medicine and "Ubshington, Pathology, University of Virginia School of Medicine, Medicine. Charlottesville
To determine whether rhinovirus infection induced a systemic cellular immune response in humans, specific antigen-stimulated blastogenesis, natural killer cell activity, and mitogenstimulated production of interleukin-2 and interferon--y by peripheral blood mononuclear cells (pBMC) (PBMC) were quantified during experimental rhinovirus infection of normal volunteers. Phytohemagglutinin-stimulated interleukin-2 production by PBMC collected on day 5 after rhinovirus inoculation was increased fourfold compared with production by PBMC collected before rhinovirus challenge (P < .05); phytohemagglutinin-stimulated interferon--y interferon-)' production was doubled (P < .05). An inverse relationship was observed between the increase in interleukin-2 production and both nasal mucus production (P < .02) and the number of days virus was cultured from nasal washings (P < .02). Natural killer cell-mediated cytotoxicity of PBMC collected on day nasal 5 after rhinovirus challenge was also increased (P < .01) compared with preinfection levels, as was specific antigen-stimulated blastogenesis on day 21 (P < .05). The extent of blastogenic response correlated directly with both mucus production (P < .05) and the number of days virus was cultured from nasal washings (P < .05). These observations are consistent with the hypothesis that rhinovirus infection results in activation of a systemic cellular immune response. Upper respiratory tract infections are the most common rhinoviruse~ are responsible for of human viral illnesses, and rhinoviruses. [1, 2]. Rhinovirus colds are 30 % of these infections [I, at least 30% prejumed pre~umed to be local rather than systemic infections; the immune responses responsible for successful host defense against this agent have not been fully defined. Local and systemic neutralizing antibody is usually not detectable until after resolution of illness, suggesting that alternate host defense mechanisms combat acute infection. Because of acute decreases in peripheral blood T lymphocyte counts during rhinovirus infection, it has been postulated that lymphocyte subsets might migrate to the nasal epithelium to provide in
Received 18 August 1989; revised 2 April 1990. Informed consent was obtained from all study subjects; guidelines of the George Washington University Medical Center Human Research Committee and the University of Virginia Human Investigation Committee were followed. Grant support: National Institutes of Health (CA-24974 to A. L. G., CA42219 to M. B. S., and HL-41507 to 1. H.), Aspirin Foundation of America, and Richardson-Vicks USA. 1. J. H. is a Pfizer Scholar. Reprints and correspondence: Dr. Judith Hsia, Department of Medicine, the George Washington University, 2150 Pennsylvania Ave., NW, WashingWashing, ton, DC 20037. * Present address: Center for Vaccine Development, University of Maryland, Baltimore. The Journal of Infectious DIseases Diseases 1990;162:591-597 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6203-0003$01.00
situ immune protection [3]. Whether these cells would exert antiviral effects through cytokine production, cytotoxic activity, or other means remains to be established. It also has been suggested that immune responses may contribute to symptoms associated with infection, since some of the side effects of treatment with recombinant interferons mimic cold symptoms [4]. To characterize the systemic cellular immune responses during rhinovirus infection and the relationship of those responses to clinical manifestations of infection, we evaluated mitogeninduced production of interleukin-2 and interferon-')' interferon--y by peripheral blood mononuclear cells (PBMC) collected during experimental rhinovirus colds in healthy adult volunteers. Natural killer cell-mediated cytotoxicity and blastogenic response ofPBMCto of PBMC to specific rhinovirus antigens were also assessed, and the relationship between changes in PBMC function and cold symptoms, mucus production, virus shedding, and antibody titers was determined.
Methods Subjects and virus infection. Healthy adult volunteers with titers of serum neutralizing antibody to an unnumbered rhinovirus sub~1:2 were recruited for participation in two clinical type (Hanks) of of~1:2 trials. Individuals with upper respiratory tract illnesses within 2 weeks or who were concurrently taking any medications were excluded. Volunteers were housed individually in motel rooms for 5 days be-
592
Hsia et aI.
ginning on the day of viral challenge (day 1 of the study). An unnumbered rhinovirus subtype (Hanks) was administered by intranasal drops (OJ (0.1 ml/nostril); the total inoculum was "'200 f\J200 50% tissue culture infectious doses (TCIDso) per subject. In the first trial, 20 volunteers were randomly assigned to receive oral aspirin, 325 mg (Miles Laboratories, Washington, DC), or placebo every other day for three doses beginning 24 h before virus challenge. In the second study 20 volunteers were randomized to receive another nonsteroidal antiinflammatory agent or placebo. In this report, only results obtained from the placebo recipients in these trials are included. Surveillance and sampling. Clinical symptoms were monitored during the 5 days after viral challenge, and nasal secretions were weighed by previously described methods [5, 6]. The occurrence of colds was detennined determined by previously described modifications [5, 6] of the method of Jackson et al. [7]. Rates of infection were determined by virus isolation from nasal washings and by measurements of serum neutralizing antibody in paired specimens obtained on the day before viral challenge and 3 weeks later. A fourfold rise in sewas accepted as evirum titer of homotypic neutralizing antibody 'WaS dence of rhinovirus infection. In the first study, sera and heparinized blood samples were collected before and 5 days after virus inoculadetermining serum interferon-')' levels and isolating PBMC tion for detennining on lymphocyte separation medium (Organon Teknika, West Chester, PA) [8]. Heparinized blood samples were also collected 21 days after viral challenge. In the second trial, heparinized blood samples for PBMC isolation were collected before virus inoculation (day 1) and on days 3 and 5. Assays on samples from individual volunteers were run concurrently. Cytokine assays. PBMC were washed, adjusted to 107 cells/ml, (l°C/min) in RPMI 1640 medium (GffiCO, (GmCO, Grand Israte-frozen OOC/min) l1g/ml gentamiland, NY) with 25 mM HEPES buffer, pH 7.5, 10 flglml (HRPMI) supplemented with 60% human cin, and 2 mM glutamine (HRPMl) AB serum·and 20% dimethyl sulfoxide (Fisher Scientific, Columbia, MD) in a linear rate freezer to -70°C, and stored in liquid nitrogen. For immunoassays, frozen PBMC (1 ml) were gently agitated in a water bath (40°C) until just melted. After addition of 1I ml of 20% human serum in HRPMI and incubation (10 min at room temperature), PBMC were transferred to 15-ml conical tubes and incubated with an additional 10 ml of 20% serum (15 min at room temperature). Cells were then washed twice in HRPMI for subsequent cytokine, natural killer cell activity, and blastogenesis assays. determined by trypan blue exclusion, was 85%-90%. Viability, detennined interferon-')' experiments, duplicate samples of PBMC (0.5 For interferon.'Y x 1()6 cells/ml) were incubated in HRPMI with phytohemaggluti#A8!ml, nin (Burroughs Wellcome, Research Triangle Park, NC), 1 pglml, and 1% human AB serum in 96-well flat-bottomed microtiter plates (Nunc, Kamstrup, Denmark) at 37°C in 5% C02: 95% humidified To determine whether cytokine production had been maximally air. Th J.tg/ml (Alpha One Biostimulated, thymosin fraction 5 (TF5), 300 JLg/ml medicals, Washington, DC), a partially purified calf thymic preparation [9], was added to some wells after 48 h. Supernatants were collected at 72 h and stored (-20°C) for interferon-')' interferon-'Y radioimmunoassay (Centocor, Malvern, PA). For interleukin-2 experiments, samples of PBMC (106 cells/ml) J.tg/ml, and were incubated in HRPMI with phytohemagglutinin, 2 JLg/ml, 1% AB serum with or without TF5 in 24-well tissue culture plates 1% (Costar, Cambridge, MA) for 24 h. PBMC were removed by centrifugation (800 g, 12 min at room temperature), and supernatants
lID 1990;162 (September)
interleukin-2-. >-
00
0
CI:l
0
0
..
~ :i'
.E oS
