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Interleukin-l-Inhibitor Activity Induced by Respiratory Syncytial Virus: Abrogation of Virus-Specific and Alternate Human Lymphocyte Proliferative Responses Alan R. Salkind, Donna O. McCarthy,· Joan E. Nichols, Frank M. Domurat, Edward E. Walsh, and Norbert J. Roberts, Jr.

Infectious Diseases Unit, Department of Medicine, School of Medicine, and School of Nursing, University of Rochester, New York

Respiratory syncytial virus (RSV) has been shown to be an important respiratory pathogen for infants and young children [1-3]. In adults it can cause illness that is clinically indistinguishable from that caused by influenza virus [4]. Reinfection with RSV is common despite limited evidence of antigenic shift or drift of the infecting virus and despite serologic evidence of host recognition of the challenging strain [2, 3, 5, 6]. Second infections by RSV result in enhanced antibody responses [7, 8], indicating previous immunologic sensitization, but occur nonetheless. In fact, volunteers can be infected repeatedly with the same isolate of RSV [9]. In contrast, infection with influenza virus commonly results in immunity to the infecting strain, although reinfections with homotypic strains have been reported [10]. We have shown previously that supernatant culture fluids derived from monocytes/macrophages exposed to either influenza virus or RSV contain both interleukin-I (IL-I) and

Received 6 March 1990; revised 30 July 1990. Presented in part: National Meetings of the American Federation for Clinical Research (San Diego, 1987) and Society for Leukocyte Biology (Marco Island, FL, 1989). Informed consent was obtained from all subjects, and studies were approved by the Committee on Use of Human Subjects of the University of Rochester Medical Center. Grant support: National Institutes of Health (AI-15547, AI-23774) and Robert WoodJohnson Clinical Nurse Scholars Program (fellowship to D.O.M). Reprints or correspondence: Dr. Norbert 1. Roberts, Jr., Infectious Diseases Unit, Box 689, University of Rochester School of Medicine, Rochester, NY 14642. * Present address: School of Nursing, University of Wisconsin Center for Health Sciences, Madison. The Journal or Infectious Diseases 1991;163:71-77 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6301-0013$01.00

IL-I-inhibitor activity [II]. However, the fluids derived from the influenza virus-exposed cells contained net IL-I activity while those from RSV-exposed cells had net IL-I-inhibitor activity, as demonstrated using the standard mouse thymocyte [11] or human mononuclear leukocyte (MNL) [12] comitogen proliferation assay. These data raised the possibility that RSV-induced inhibition ofIL-I activity could play a role in the abrogation of an early, anamnestic immune response, permitting the recurrence of clinically evident RSV infection by inhibiting the proliferation of virus-specific lymphocytes in early stages of the challenge. The current studies were done to examine lymphocyte activation markers and cell cycle profiles after exposure to RSV to detect the anticipated consequences ofIL-I-inhibitor activity, to determine the role of viral infectivity in eliciting IL-Iinhibitor production or in suppressing mitogen- and nonviral antigen-induced proliferation, and to examine whether antiRSV proliferative responses specifically are abrogated by the observed net IL-I-inhibitor activity.

Materials and Methods Cellcollection and exposure to viruses. Standard techniques were used to produce and measure viral innocula. RSV (Long strain, ATCC) was grown in HEp-2 cells, and influenza A/AA/Marton/43 HINI was grown in allantoic cavities of lO-day-oldembryonated hen's eggs [12, 13]. Each virus was stored at -70°C until used. Earlier studies showed that neither HEp-2 culture fluid nor allantoic fluid (in the absence of virus) affect cell responses [13]. Recent preliminary experiments also established that the effects of the viruses in the current studies could not be attributed to either HEp-2 culture fluid or allantoic fluid. Such experiments specifically showed no effect of the control fluids for each virus on thymidine incorporation by the thymocytes (IL-I assay), leukocyte expression of markers such

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Respiratory syncytial virus (RSV) infection has been shown to induce human mononuclear leukocyte (MNL) production of net interleukin-l (IL-l)-inhibitor activity. In the current studies ofIL-l-inhibitor effects, RSV-exposed cellswerecompared with autologousMNLthat wereshamexposed or exposed to inactivated RSV or influenza virus (which induces net IL-l activity and commonly elicits effective homotypic immunity). Exposure ofMNL to influenza virus or inactivated RSV resulted in increased expressionof human leukocyte antigen-DR, the IL-2 receptor, and the transferrin receptor and increased progression through the cell cycleby 3 days. In contrast, exposure to infectious RSV resulted in decreased marker expressionand cell cycle arrest, with abrogation of proliferation in response to the virus or other stimuli. These data raise the possibility that a contributing mechanism for recurrence of RSV infection is early suppression of the clonal expansion of virus-specific lymphocytes due to net IL-l-inhibitor activity.

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Salkind et al.

Exposure of purified monocytes/macrophages to RSV proteins.

The major surface glycoprotein and fusion protein of RSV were purified by immunoaffinity chromatography as describedpreviously [17, 18]. Purified monocytes/macrophages wereobtainedfrom MNL by washingculture dishes extensively after 24 h to remove nonadherentcells,followed by collection of the adherent rnacrophages using a rubber policeman [14]. Aliquots of the monocytes/macrophages were then sham-exposed (using the buffer for the purifiedproteins) or exposed to theproteins at concentrations ranging from 1.0to 0.001 mg/rnl. After 1 h, the cells were washedand cultured for a further 24 h. Cell-freesupernatants were then collectedand assayedfor ILl and IL-1-inhibitoractivity. Resultsare shownfor cultures stimulated with 1 Jotg/rnl, which are representative of overall results. Analysis of expression of cell surface markers and cell cycle profiles. Expressionof surface markers indicative of activationor

proliferation wasmeasured usingdual-eolor immunofluorescent labeling and flow cytometry analysis[19, 20). MNL weresham-exposed, exposed to RSV, or exposed to inactivatedRSV or influenzavirus and incubatedfor 72 h as described above. After incubation, MNL were washedand stained with phycoerythrin-labeled anti Leu M3, which reactswith maturemonocytesand macrophages, followed by fluorescein-labeled anti-human HLA-DR, IL-2 receptor (IL-2R), or transferrin receptor (TFR) (Becton-Dickinson, Mountain View, CA). Stained MNL were analyzed using a Becton-Dickinson FACScan flow cytometer. Cell cycle profiles were obtained by labeling MNL with bromodeoxyuridine (BrdU) followed by stainingwithpropidium iodideand anti-BrdU [21]. Cell labelingwas assessedby flow cytometry using Becton-Dickinson DNA software. Standard thymocyte lL-1 and Il-l-inhibitor assay. Mousethymocytes were obtainedfrom 6- to 8-week-old C3H/HeJ mice and suspended at 5 x 106 cells/ml in medium supplemented with 5% heat-inactivated FCS, 10 rnM HEPES buffer, 2.5 x 10-5 M 2-mercaptoethanol, andpenicillin,as describedpreviously [11]. The cells wereplacedin microtiterplates in the presenceof 5 Jotg/rnl phytohemagglutinin (PHA-P; Difco, Detroit) and serial dilutions of supernatants collected from sham- or virus-exposed MNL. The thymocytes wereincubated for 96 h, with the addition of PH]thymidine for the last 24 h of culture. Thymocytes were harvested and incorporation of pH]thymidine was determined using a liquidscintillation counter.

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NOTE. Results of four experiments are shown. Mean percentage ± SE of control cells expressing human leukocyte antigen (HLA)-DR, the interleukin-2 receptor (IL-2R), and the transferrin receptor (TFR) were 4.45 ± 0.84, 4.79 ± 1.44, and 2.51 ± 0.76, respectively.

Each assayincludedserial dilutionsof purifiedhumanIL-1 (Genzyme, Cambridge, MA) as a reference standard for activity. Each assay alsoincluded, as controls, determination ofthymocyte responses to the inducingvirus (equivalent to cell exposure inoculum, without cell-derived factors). A 1:25 dilutionof purifiedhumanIL-l was addedto parallel plates in mousethymocyte assaysto determineILl-inhibitor activityin supernatants [11]. The viabilityofthymocytes that receivedfluidsfrom RSV-exposed MNL or from sham-exposed MNL did not differ, as determined by exclusionof trypan blue dye [14] at the end of 4 days of culture. Human MNL proliferation assay. Human MNL were obtained as described above, added to microtiter wells, and exposed to the viruses. Control, RSV-exposed, and inactivated RSV-exposed MNL were suspendedin FCS-supplemented medium and were placed in 96-well microtiterplates (5 x 106 cells/rnl) and incubated for 7 days [14,20]. The cells were pulsed with [3H]thymidine for the terminal5 h of incubation, harvested, and counted with a liquid scintillation counter. In parallelassays, autologous sham-andvirus-exposed MNL were also incubated in the microtiter plates in the presence of the mitogen PHA (20 Jotg/rnl) or the nonviralantigen streptokinase-streptodornase (SK-SD, 20 units/rnl; Lederle Laboratories, Pearl River, NY) [20]. The cells were incubated for 72 h with PHA and 7 days with SK-SD, with the addition of [3H]thymidine during the last 5 h of culture. Data analysis. Data were analyzed using analysis of variance withthe Scheffe test for multiplecomparisons.The fewstudieswith two determinations only were analyzed using the paired t test.

Results Antiproliferative effectsofIL-I-inhibitoractivity. A small (absolute) percentage oflymphocytes expressing markers associated with proliferation relative to control MNL began to appear in culture early after exposure of MNL to influenza virus (table 1). In contrast, the cultures of MNL exposed to RSV showed a decreased percentage of cells expressing proliferation markers relative to control MNL. Figure 1 provides a representative set of flowcytometry histograms showing expression of HLA-DR, the IL-2R, and the TFR. The small but completely consistent changes were apparent by 2-3 days after exposure. The absolute increase in percentage of cells expressing the markers after exposure to influenza virus was small (table 1). However, the changes represented a

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as human leukocyteantigen (HLA)-DR and the transferrin receptor, and cell cycle progression. MNL wereobtainedfromperipheralbloodof healthy adult volunteer subjects by ficoll-hypaque sedimentation [14, 15]. All adults we have tested have antibodies to RSV detectable by ELISA [13, 16]. MNL were incubated at a concentration of 5 x 106 in plastic Petri dishes at 37°C in serum-free Eagle's MEM for exposure to the viruses [14]. In all experiments, comparisons were made between aliquots of autologous MNL examined concomitantly. Aliquots of cells were sham-exposed or exposed to RSV, heat-inactivated RSV(56°C,2 h), or influenzavirus at 1 MOL After a 1-hincubation, the cells were washed extensively with PBS, resuspended in medium with 10% heat-inactivated fetalcalf serum (FCS), and incubated for 2-10 days. Cell-free supernatants from the MNL were collected after 3 days by centrifugation, aliquoted, and stored at 4°C until being assayed for IL-1 andIL-l-inhibitoractivity. The MNL werecounted andused in assaysfor surfacemarker expressionand cell cycleprogression.

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Log fluorescence intensity relative percentage increase from 65 % to 138 % over control MNL. Furthermore, in three experiments, MNL also were assayed for expression of the markers 7 days after exposure or shamexposure. By that time, MNL exposed to influenza virus showed substantial mean absolute percentage changes in expression of the markers associated with proliferation (14.5 % and 17.6% change from control MNL for IL-2R and TFR, respectively). In contrast, MNL exposed to RSV still showed no increase in percentage of cells expressing markers of proliferation (1.1 % and 1.5% change, respectively). By 7 days after exposure, there were still differences in expression of the activation marker HLA-DR, although differences between MNL exposed to influenza virus or RSV were less pronounced (15.4% vs. 8.6%). Cell cycle analysis was done 3 days after exposure or shamexposure and showed an increased percentage of cells proliferating (in the S, G2 , or M phases) after exposure to influenza virus compared with control cultures (figure 2). In contrast, after exposure to RSV, cells were largely confined to the GO/G 1 phase, showing a decreased percentage of cells entering the cell cycle relative to control cultures. Similar results were obtained using anti-BrdU monoclonal antibody labeling to determine cell cycle profiles (data not shown).

Role of infectivity in production of IL-l-inhibitor activity. The viability ofMNL exposed to RSV was not different

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major surface glycoprotein or fusion protein could induce net IL-I-inhibitor activity. Unfortunately, the mouse thymocyte bioassay for IL-I (figure 4, left) was confounded by the direct stimulation of thymocyte proliferation by the buffer used to purify the RSV-derived proteins, as well as by many other buffers tested for the purification process and bioassay. From the thymocyte cultures with added exogenous IL-I, however, it appeared that supernatant fluids obtained from monocytes/macrophages exposed to purified proteins did not contain net IL-I-inhibitor activity. The ELISA of the fluids for IL-I (figure 4, right) showed that the buffer itself did not induce IL-I production by the monocytes/rnacrophages, whereas both RSV-derived proteins were effective inducers of IL-I production. MNL also were exposed to infectious and inactivated RSV to determine whether the above-noted correlation between viral infectivity and IL-I-inhibitor production would be reflected in MNL proliferative responses to mitogen- and nonviral antigen stimulation. MNL exposed to RSV showed decreased thymidine incorporation compared with either control MNL or MNL exposed to inactivated RSV (P = .03 for PHAstimulated and P = .0001 for SK-SD-stimulated RSV-exposed MNL relative to other MNL) (figure 5).

Effects of IL-I-inhibitor activity on RSV-specific proliferation. An increased percentage of cells proliferated (during from that of sham-exposed MNL or MNL exposed to inactivated RSV, as determined by exclusion of trypan blue dye 3 days after exposure. Supernatants from the MNL exposed to RSV contained net IL-l-inhibitor activity (P = .0001 compared with fluids from other MNL), whereas the fluids derived from MNL exposed to inactivated RSV contained net IL-l activity (P < .0001 compared with fluids from other MNL; figure 3). Fluids from the RSV-exposed MNL did not contain detectable infectious virus when added to HEp-2 cells and examined for up to 14 days. We also sought to determine whether purified RSV-derived

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Previous studies demonstrated that supernatant fluids from RSV-exposed MNL contained IL-l inhibitors with antiproliferative activity as determined using thymocyte or human lymphocyte thymidine incorporation assays [11, 12]. The current data extend that observation in several significant ways. First, the data demonstrate that this anti proliferative activity can be correlated with a reduction in cell surface markers indicative of activation or proliferation, arrest of MNL progression through the cell cycle, and a decreased number of cells in culture (relative to controls) after exposure to RSV. The differences among cells exposed to infectious RSV, sham-exposed, or exposed to alternate virus preparations were detectable early (2-3 days) after challenge.

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Figure 5. Mononuclear leukocyte proliferative responses to mitogen (left) and antigen (right) after shamexposure (Control) or exposure to respiratory syncytial virus (RSV) or inactivated RSV (lnact RSV). Results are means of six experiments, each with mitogen and antigen stimulation. Unstimulated cells incorporated

Interleukin-1-inhibitor activity induced by respiratory syncytial virus: abrogation of virus-specific and alternate human lymphocyte proliferative responses.

Respiratory syncytial virus (RSV) infection has been shown to induce human mononuclear leukocyte (MNL) production of net interleukin-1 (IL-1)-inhibito...
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