avidity of ICAM- 1 and LFA- 1 would affect initiation and progression

Transcription

1 Suppressed Expression of ICAM-1 and LFA-1 and Abrogation of Leukocyte Collaboration after Exposure of Human Mononuclear Leukocytes to Respiratory Syncytial Virus In Vitro Comparison wit Exposure to Influenza Virus Alan R. Salkind, Joan E. Nicols, and Norbert J. Roberts, Jr. Infectious Diseases Unit, Department ofmedicine, University ofrocester Scool ofmedicine, Rocester, New York Abstract Human mononuclear leukocytes (MNL) exposed to respiratory syncytial virus (RSV) produce net IL-1 inibitor bioactivity wit te anticipated consequences of cell cycle arrest, suppressed virus-specific proliferation, and reduced expression of activation markers. Tese studies were undertaken to investigate effects of exposure and resultant net IL-1 inibitor activity on te expression of te intercellular adesion molecule-i (ICAM-1), and its ligand te lympocyte function-associated antigen (LFA-1). MNL collected at 1, 4, and 24 after exposure to influenza virus (wic induces net IL-1 bioactivity) sowed enanced expression of ICAM-1 and LFA-1 relative to sam-exposed MNL and exibited cell clustering. In contrast, exposure to RSV was associated wit suppressed expression of bot ICAM-1 and LFA-1 and wit minimal detectable cell clustering trougout te culture period. Influenza virus-exposed MNL produced significantly more IL-1 and IFN-'y (wic require cell-cell collaboration for optimal production) tan did RSV-exposed MNL. Tese data raise te possibility tat exposure of MNL to RSV fails to elicit or blocks te early events necessary for cellular collaboration, contributing to early suppression of te clonal expansion of RSV-specific lympocytes. (J. Clin. Invest : ) Key words: immunoregulation * interferon-gamma - interleukin-1 - lympocyte function associated antigen-i * intercellular adesion molecule-i Introduction Cell-cell interaction between antigen presenting cells (APC)' and T lympocytes is an essential process in te generation of Tis work was presented at te 12t International RES Congress and 27t Annual Meeting ofte Society for Leukocyte Biology, Heraklion, Crete, Greece, October Address correspondence and reprint requests to N. J. Roberts, Jr., Infectious Disease Unit, Box 689, University of Rocester Scool of Medicine, Rocester, NY Receivedfor publication 10 January 1991 and in revisedform 11 April Abbreviations used in tis paper: APC, antigen presenting cells; ICAM- 1, intracellular adesion molecule- 1; LFA- 1, lympocyte function-associated antigen- 1; MNL, mononuclear leukocytes; MOI, multiplicity of infection; RSV, respiratory syncytial virus. J. Clin. Invest. Te American Society for Clinical Investigation, Inc /91/08/0505/07 $2.00 Volume 88, August 1991, immunological responses (1-4). Tis interaction is tougt to consist of bot a specific signal, namely recognition of te MHC-antigen complex and te occupied T cell receptor (5, 6), and nonspecific or accessory signals, suc as expression of costimulatory molecules or production of IL-1 (7, 8). Te complete repertoire of accessory signals is yet to be establised. Noneteless, factors tat interfere wit eiter specific signals or accessory signals may be expected to result in an abrogated immune response (9). Several adesion molecules ave been identified tat are tougt to play a central role in te interaction ofte APC wit te T lympocyte (10). Intercellular adesion molecule-1 (ICAM- 1) serves as te major ligand for lympocyte functionassociated antigen- 1 (LFA- 1) (1 1, 12). Te expression and/or avidity oficam-l is augmented upon cell activation and in te presence of inflammatory cytokines, suc as IL- 1 and IFN--y ( 13). In contrast, te expression oflfa- I as not been demonstrated to be cytokine sensitive (14). However, LFA- 1 is tougt to increase in expression and/or avidity for ICAM- 1 upon cell activation (15). Complete T lympocyte activation, demonstrated by IL-2 production and DNA syntesis, as been sown to occur wit simultaneous cross-linking oflfa- 1 and CD3 (16). Terefore, modulation ofte expression and/or avidity of ICAM- 1 and LFA- 1 would affect initiation and progression of te immune response. We ave sown previously tat uman mononuclear leukocytes (MNL) tat ave been exposed to respiratory syncytial virus (RSV) produce net IL- 1 inibitor bioactivity (17, 18). Te anticipated consequences of suc net IL- 1 inibition were demonstrated: namely, cell cycle arrest, suppressed proliferation, and a decrease in te expression of cell surface markers indicative of activation and/or proliferation (19). Tese observations suggested tat net IL- 1 inibitor activity could result in abrogation or delay of RSV-specific anamnestic immune responses by inibiting te early clonal expansion of RSV-specific lympocytes. Te well establised ability of RSV to reinfect individuals wo ave preexisting cellular and umoral evidence of immunity to te virus (20-22) may represent an in vivo correlate of suc in vitro observations. Tese studies were undertaken to determine weter exposure to RSV and induction of net IL- I inibitor activity affects te early collaborative events leading to emergence of RSVspecific MNL responses, events tat include expression of ICAM- 1 and/or LFA-1 and leukocyte clustering. Te studies also examined production of te cytokines IL-1 and IFN-'y, wic require cell-cell collaboration for optimal production (23, 24). In all experiments, autologous MNL were sam exposed or exposed to influenza virus (wic induces net IL-l bioactivity) for comparison. Adesion Molecules and Respiratory Syncytial Virus 505

2 Metods Cell collection and exposure to viruses. Standard tecniques were used to produce and measure viral inocula. RSV (Long strain; American Type Culture Collection, Rockville, MD) was grown in HEp-2 cells, and influenza A/AA/Marton/43 HINI was grown in allantoic cavities of 10-d-old embryonated en's eggs (17, 25). Eac virus was stored at -70'C until used. Bot RSV and influenza virus pools titered 108 plaque forming units/ml using HEp-2 cells or MDCK cells in assays, respectively (25). Previous studies sowed tat neiter HEp-2 culture fluid nor allantoic fluid affect te proliferative response or te expression of activation markers by MNL (19). Periperal blood collected from ealty adult donors was separated by Ficoll-Hypaque sedimentation (26). In all experiments, comparisons were made between aliquots ofautologous cells examined concomitantly. Aliquots of cells were sam-exposed at 370C for 1 in serumfree Eagle's minimum essential medium or exposed to influenza virus or RSV at a multiplicity of infection (MOI) of one (17-19). Previous studies ave sown tat, at equal MOI, te percentage ofmnl infected by influenza virus or RSV is relatively similar (27, 28) (Roberts, N. J., Jr., and J. E. Nicols, unpublised observations). Te MNL cultures were ten supplemented wit 10% eat-inactivated FCS and incubated for 1, 4, 24, and 72 before analysis. Cell-free supernatant fluid was collected by centrifugation at 24 and 72 after exposure for analysis of IL-l and IFN-y activity. Cells were collected at 1, 4, and 24 for analysis by flow cytometry or, in oter experiments described below, were stained in situ and examined by ligt microscopy. For all experiments, viability was assayed by trypan blue exclusion. Viability (always > 95%) was similar after exposure to te different viruses. IL-I# and IFN-'y assays. Supernatant fluids were obtained from sam-exposed, influenza virus-infected or RSV-infected MNL as described above. Cell-free fluids were collected at 1 and 3 days after infection and stored at -70'C until assayed. Quantitative measurement of IL-, I, wic is te soluble form produced by monocytes-macropages after activation (29), was obtained by ELISA (Cistron, Pine Brook, NJ). IFN-'y was measured using a solid pase radioimmunoassay (Centocor, Malvern, PA). Microscopic analysis. Te extent of cell clustering after exposure was determined by culturing cells over plastic cover slips. Cover slips from te cultures were retrieved at 1, 4, and 24 after sam-exposure or exposure to influenza virus or RSV. Cells were stained by te Diff- Quick metod (Dade Diagnostics, Aquada, PR) immediately after collection of coverslips and examined for cell clustering using a Zeiss microscope. Analysis of expression of cell adesion molecules. Expression of cellular adesion molecules was measured using indirect immunofluorescent labelling and flow cytometry. MNL were also analyzed by fluorescence microscopy to verify tat fluorescence was associated wit a cell-surface pattern of staining. MNL were eiter sam-exposed or exposed to influenza virus or RSV. After furter incubation, te live MNL were wased and stained wit eiter murine monoclonal anti- ICAM-l or anti-lfa-l (AMAC, Inc., Westbrook, ME) at 40C for 30 min. Te cells were ten wased and stained at 40C for 30 min wit FITC-conjugated goat anti-mouse antibody (Tago, Inc., Burlingame, CA) (13, 30). Analysis of stained MNL (monocytes-macropages and lympocytes) was performed on a Becton-Dickinson FACScan" flow cytometer (Mountain View, CA) by gating on te respective populations using forward and side ligt scatter (28) (Fig. 1 a). In all experiments, te cursor for te negative population of cells was set using a mouse isotype (IgGj) control monoclonal antibody (Becton-Dickinson). Representative istograms (and cursor settings) for control MNL are sown in Fig. 1, b-e and described in Results. Data analysis. Results represent te mean±sem for five or more experiments. Determination of significant differences (paired t test) was performed using te commercially available software program Faststat (Systat, Inc., Evanston, IL). zm =.- Pa Go 4) E; a) d) a] 1* 'I&-, z-t&- Fluorescence Intensity Figure 1. Cell population gatings for flow cytometry analyses. Control MNL are sown. (a) Monocyte-macropage and lympocyte gates using forward and side ligt scatter caracterization: 1, monocytes-macropages; 2, lympocytes. (b-e) Histograms of monocytes-macropages (b and c) and lympocytes (d and e) stained for expression of ICAM- I (b and d) and LFA- I (c and e). Te clear istograms depict isotype control antibody staining. Te solid istograms depict staining wit te respective murine monoclonal anti- ICAM- I and anti-lfa- I antibodies. Negative (nonstained), "dim" (intermediate staining, except d), and "brigt" (intensely stained) populations are separated by te cursors. Results Microscopic analysis. Cell-cell interaction is a sentinel event in te induction and progression of te immunological response. Te formation of cellular clusters was examined at 1, 4, and 24 after uman MNL were sam-exposed or exposed to influenza virus or RSV. As early as 1, and extending troug te period of culture, MNL exposed to influenza virus sowed increased cluster formation (Fig. 2). In contrast, sam-exposed MNL or MNL exposed to RSV sowed minimal clustering. Cytokine production. Earlier studies sowed tat RSV-exposed MNL did not proliferate in response to te virus (17, 19). Te observation tat RSV-exposed MNL sowed a diminised or absent clustering pattern prompted us to investigate te production of IL- I and IFN-'y, bot of wic require cellular collaboration (but not cellular proliferation) for optimal expression (23, 24, 31). Determination of IL-lI3 activity demonstrated tat tere was a minimal amount of IL-I produced by control cells by 24 and 72 (Fig. 3). Bot influenza virus and 506 A. R. Salkind, J. E. Nicols, and N. J. Roberts, Jr.

3 a fe $ +,.;;{. s, t Z. v. os A.. ds. 4 > \ b \. A, g c we a 4060 d * I,,., a e, _, As *., fi as k I,, a. Figure 2. Cell cultures stained for analysis of cell cluster formation. Cover slips from MNL cultures were stained 1 (a-d), 4 (e-), and 24 (i-i) after sam exposure (Control; a, e, i) or exposure to influenza virus (b, f j and c, g, k) or RSV (d,, 1). Representative IOX potomicrograp fields are sown, wit potomicrograps c, g, and k sowing clusters from influenza virus-exposed cultures viewed under oil at IOOX. RSV induced IL- I production by 24, wit a small difference (P = 0.058) in te magnitude of response to te viruses. In contrast, by 3 d after exposure te quantity ofil-i produced by influenza virus-exposed cells continued to increase (P = 0.026), wile tat of RSV-exposed cells decreased to a level below tat of day 1. Te production of IFN-y by MNL exposed to influenza virus was substantial by one day in culture (P = and versus control and RSV-exposed MNL, respectively) (Fig. 4). MNL tat were sam-exposed or exposed to RSV produced minimal IFN-?y. 3 d after exposure, fluids from sam-exposed cells sowed little additional accumulation ofifn-y, but IFN-"y was produced by MNL in response to RSV (P = 0.005) or influenza virus (P = 0.008). Altoug IFN-'y was detectable in fluids from RSV-exposed cells by tree days in culture, te titers were still several-fold lower tan tose produced by cells exposed to influenza virus (P = 0.050). Cell surface expression oficam-j and LFA-i. Te above and earlier studies suggested tat cell collaboration, essential for bot cytokine production and lympocyte proliferative responses, is suppressed at an early stage after exposure of MNL to RSV. Alteration of te expression and/or avidity of ICAM- 1/LFA- 1 by RSV-exposed MNL could be one mecanism contributing to diminised leukocyte clustering and collaboration, and expression of tese molecules was examined. Cells were exposed and cultured as MNL, wit separate analysis by appropriate gating (Fig. 1) during flow cytometry. Analysis of MNL by fluorescence microscopy sowed a rim offluorescence indicative ofcell surface staining. Profiles oficam-l/lfa-l expression by control MNL were similar in regard to intensity and percentage of positive cells to tose of previous reports (30, 32, 33) (see Fig. 1), and suc results were compared wit profiles of virus-exposed MNL. Expression oficam- 1 by monocytes-macropages sowed tat tree monocyte-macropage populations could be identified based on fluorescence intensity. A fluorescence-negative population, a population wit intermediate (dim) fluorescence intensity, and a brigtly stained population of monocyte-ma E Qa CCr. z:i w= 500- w I- nil 1 3 DAYS AFTER EXPOSUI Figure 3. Interleukin-l # production by control MNL (solid bars), influenza virus-exposed MNL (open bars), and RSV-exposed MNL,552 (atced bars). Results represent mean RE titers±sem. 2 IL a: I I DAYS AFTER EXPOS Figure 4. Interferon--y production by control MNL (solid bars), influenza virus-exposed MNL (open bars), and RSV-exposed MNL (atced bars). Results 3 represent mean 'URE titers±sem. Adesion Molecules and Respiratory Syncytial Virus 507

4 cropages were identified after staining for ICAM- 1 (Fig. 1 b). An increased percentage of monocytes-macropages exposed to influenza virus stained in te fluorescence-negative and, to a greater extent, te "brigt" range for ICAM-l trougout te culture period (Table I). Conversely, a decreased percentage stained in te "dim" range. In contrast, monocytes-macropages tat were eiter sam-exposed or exposed to RSV sowed only minor sifts in te cell populations over te duration of culture (Table I). Te redistribution of monocytesmacropages exposed to influenza virus from te "dim" to "brigt" population was a small (absolute) percentage at 1 and 4 relative to eiter sam-exposed or RSV-exposed cells, but te "brigt" population increased substantially by 24 after exposure (P = and compared wit control or RSVexposed monocytes-macropages, respectively). In comparison, tose cells tat were eiter sam-exposed or exposed to RSV ad a smaller percentage of cells staining wit a "brigt" fluorescent intensity at 24 compared wit 4 after exposure. Te assay for expression of ICAM-I on lympocytes sowed tat exposed cells could be divided into a negative and positive staining population on te basis of te IgG(1) isotype control. In some experiments, a tird population of cells wit intermediate fluorescent intensity was detected (for example see Fig. 1 d). However, tis was an inconsistent finding, and tis intermediate subset ofcells was terefore not separated for analysis. At 1 and 4 after exposure to influenza virus, tere was an absolute but small increase in te number of lympocytes expressing ICAM- I relative to eiter te control or RSVexposed cells (Table II). By 24, owever, tere was a large increase in te percentage of influenza virus-exposed lympocytes expressing ICAM-l (P = and compared wit control and RSV-exposed lympocytes, respectively). In contrast, lympocytes exposed to RSV sowed a minimal increase in teir expression of ICAM- I from 1 to 4 in culture. From 4 to 24 after exposure tere was no appreciable cange in te expression of ICAM- I by RSV-exposed lympocytes. At 24 of culture, expression of ICAM-I was sligtly below tat observed wit sam-exposed cells. Analysis of te monocyte-macropage population for ex- Table I. Expression oficam-j by Monocytes-Macropages* Time after Fluorescence exposure intensity Control Influenza virus RSV 1 Negative 3.49± ± ±0.69 Dim 91.62± ± ±1.60 Brigt 4.89± ± ± Negative 3.29± ± ±1.05 Dim 92.12± ± ±2.87 Brigt 4.59± ± ± Negative 3.43± ± ±1.23 Dim 92.90± ±5.86* 92.46±1.59 Brigt 3.67± ± ±0.71 * Data are expressed as te mean percentage±sem of cells wit te described staining for ICAM- 1. tp = and 0.021, and I P = and compared wit control cells and cells exposed to RSV, respectively. Table I. Expression oficam-i by Lympocytes* Time after Fluorescence exposure intensity Control Influenza virus RSV I Negative 70.01± ± ±2.70 Brigt 29.99± ± ± Negative 73.49± ± ±3.42 Brigt 26.51± ± ± Negative 68.42± ± ±6.52 Brigt 31.58± ±9.93* 30.91±6.52 * Data are expressed as te mean percentage±sem of cells wit te described staining for ICAM-1. $ P = and compared wit control cells and cells exposed to RSV, respectively. pression of LFA- 1 sowed an antigenic profile similar to tat seen wit ICAM- 1 expression by monocytes-macropages (Fig. 1 c). Monocytes-macropages exposed to influenza virus demonstrated redistribution from populations of negative and "dim" fluorescence intensity to a population of "brigt" fluorescence intensity (Table III). At 4 after exposure to influenza virus, te percentage of "dim" cells was significantly decreased compared wit te control population (P = 0.014). Simultaneously te number of monocyte-macropages wit "brigt" fluorescence intensity was increased compared wit control (P = 0.006) and RSV-exposed cells (P = 0.035). Examination of influenza virus-exposed monocytes-macropages after 24 sowed tat redistribution of cells into te "brigt" range ad remained stable from 4 to 24 of culture (P = and compared wit control and RSV-exposed cells, respectively). Tere was a concomitant decrease in te percentage of cells in te fluorescence-negative and "dim" ranges (P = and compared wit control and RSV-exposed monocytesmacropages, respectively) (Table III). RSV-exposed cells did not differ from control cells in expression of LFA- 1. Staining for expression of LFA- I on lympocytes sowed a Table III. Expression oflfa-1 by Monocytes-Macropages* Time after exposure Fluorescence intensity Control Influenza virus RSV 1 Negative 3.89± ± ±1.08 Dim 93.49± ± ±1.88 Brigt 2.62± ± ± Negative 3.31± ± ±1.29 Dim 91.34± ±3.24$ 90.52±3.25 Brigt 5.35± ± ± Negative 2.21± ± ±1.19 Dim 93.94± ± ±1.72 Brigt 3.85± ± ±1.21 * Data are expressed as te mean percentage±sem of cells wit te described staining for LFA- 1. $ P = and , 1 P = and 0.035, "lp = and 0.032, and ' P = and 0.03 compared wit control cells and cells exposed to RSV, respectively. 508 A. R. Salkind, J. E. Nicols, and N. J. Roberts, Jr.

5 bimodal distribution (Fig. 1 e). 1 after exposure, tere was an increase in te percentage of influenza virus-exposed lympocytes in te "brigt" population, and a concomitant decrease in te "dim" population, in comparison wit control and RSV-exposed cells (Table IV). 4 after exposure, bot te influenza virus-exposed and RSV-exposed lympocytes sowed movement ofcells from te fluorescence-negative and "brigt" populations into te "dim" population. Noneteless, influenza virus-exposed lympocytes continued to sow a relative increased number oflympocytes in te "brigt" region. Control lympocytes sowed a sligt increase in te percentage ofnegative cells along wit a decrease in LFA-l "brigt" cells and a larger percentage of cells in te LFA- 1 "dim" region wit time in culture (P = compared wit influenza virus-exposed cells). After 24 of culture, tere was a redistribution of influenza virus-exposed lympocytes from te LFA-l "dim" (P = and compared wit control and RSV-exposed lympocytes, respectively) to te LFA-l "brigt" (P = and compared wit control and RSV-exposed cells, respectively) range, wit only a minor sift ofrsv-exposed lympocytes. To eliminate te possibility tat increasing fluorescence intensity was due to an increase in cell size rater tan increased density of expression ofte molecules, cells were analyzed furter by forward ligt scatter. At tese early times after exposure, tere were no significant differences in size between cells tat were sam-exposed, exposed to influenza virus, or exposed to RSV (data not sown). Discussion Tese studies allow us to extend our understanding of te immunopatogenesis of recurrent RSV infection wit several potentially important observations. (a) Suppression of ICAM- 1/ LFA- 1 expression by MNL was evident witin 1 after exposure to RSV (relative to MNL exposed to influenza virus in parallel experiments) and was maintained trougout te culture period; (b) ICAM-l/LFA-l expression was correlated wit te extent of leukocyte cluster formation; and (c) decreased Table IV. Expression oflfa-i by Lympocytes* Time after Fluorescence exposure intensity Control Influenza virus RSV 1 Negative 1.10± ± ±0.76 Dim 50.21± ± ±5.37 Brigt 48.69± ± ± Negative 1.26± ± ±0.25 Dim 70.81± ±5.03* 63.82±7.22 Brigt 27.93± ± ± Negative 0.97± ± ±0.62 Dim 65.74± ± ±4.85 Brigt 33.29± ± ±4.67 * Data are expressed as te mean percentage±sem of cells wit te described staining for LFA- 1. t P = compared wit control cells. I P = and 0.008, and 'P = and compared wit control cells and cells exposed to RSV, respectively. ICAM-l/LFA-l expression and cell clustering were associated wit curtailed production of IL- If and IFN-'y. Te alterations in expression of ICAM-l/LFA-l by 1 were not large. However, tey could be correlated wit canges in leukocyte cluster formation (Fig. 2). Tese data suggest tat minor canges in te expression of ICAM-l/LFA-l are associated wit pysiologically relevant canges in adesion. Te kinetics and vitality of APC-lympocyte interaction after in vitro or in vivo viral callenge are not fully understood. Generation of an immune response does appear to require antigen-independent and antigen-dependent adesion between te APC and T lympocyte (10). Exposure of MNL to influenza virus resulted in an increased population of cells staining for "brigt" fluorescence intensity of ICAM-1/LFA-I after 1 in culture, a time tat may represent mainly antigen-independent leukocyte adesion. Fluorescence intensity was maximal at 24 after exposure, a time tat may represent antigen-dependent adesion. In contrast, RSV-exposed MNL did not sow an increase in te population of MNL staining for "brigt" expression of ICAM-l/LFA-l during te entire culture period relative to control MNL. Overall, te level of expression of ICAM- I/LFA- I at 1 after in vitro exposure to influenza virus or RSV was predictive ofte magnitude of expression and cellcell interaction (as demonstrated by cell cluster formation) during te remainder of te culture period. Te mecanisms tat contribute to te leukocyte responses described in tis report are not known. Production of IL-l inibitor by MNL and abrogation of collaborative leukocyte responses appear to be associated wit exposure of MNL to infectious but not inactivated RSV (19). MNL exposed to inactivated RSV produce net IL-l activity and proliferate, just as do MNL exposed to infectious or inactivated influenza virus (17, 19, 34). Influenza virus infection of periperal blood MNL appears to be abortive, wit production of viral proteins but witout release of free progeny virus (2, 28, 35). Te occasional detection of RSV in culture fluids as been associated wit MNL subpopulations tat did not produce net IL- 1 inibitor activity in our studies, and not wit monocyte-macropage or lympocyte populations tat produced te inibitory factor, even toug producer and nonproducer populations of cells were bot infected as determined by syntesis and expression of RSV F and G proteins (17, 27). Te differences between responses of MNL to influenza virus and RSV ave been observed over a wide range of MOI, including MOI tat would ensure tat virtually all cells could be infected if susceptible. Te data from immunofluorescent staining suggest tat relatively similar proportions of monocyte-macropages and lympocytes are infected by te two viruses (27, 28; Roberts, N. J., Jr., and J. E. Nicols, unpublised data), but do not establis weter te same subsets of cells are infected. Te differences in production of IL- and IL- I inibitors, and in cell-cell collaboration, are evident as early as syntesis and expression of influenza virus proteins are detected (2) and long before syntesis and expression of RSV proteins are detected (27). Te differences in production of cytokines and cell-cell interactions persist even after RSV protein syntesis and expression are clearly detected, and after influenza virus protein syntesis is no longer detectable (2, 17, 28). Te differences in response are so rapidly apparent tat tey are unlikely to be due to any delayed effect of RSV on ost cell functions, altoug suc effects also could exist. Adesion Molecules and Respiratory Syncytial Virus 509

6 Te current data raise te possibility tat early and sustained suppression of ICAM- I/LFA- I expression and/or avidity by RSV-exposed MNL is a critical mecanism wereby exposure to te virus alters te kinetics, or abrogates te vigor, of an anticipated antiviral anamnestic immune response. In vitro evidence ofte pysiological consequences ofsuppressed leukocyte cluster formation may be provided by te results of IL-I#3 and IFN-'y assays in tese studies, and te results of virus-specific and alternative lympocyte proliferation assays reported earlier (17-19). Antigen-specific lympocyte proliferation requires adequate antigen presentation and te presence of IL-1 (7, 36). Tese studies also sowed tat te production of IL-I,3 by RSV-exposed MNL is relatively reduced over 3 d of culture (Fig. 3). In addition, as sown previously (17-19), IL- I bioactivity was minimal or absent, reflecting concomitant (and excess) production of IL- I inibitor (data not sown). Te level of IL- I activity in fluids from RSV-exposed MNL at day tree was 50% of te level present at day one, possibly reflecting te balance between production and utilization. In contrast, IL-i activity was several-fold greater at day tree compared wit day one for influenza virus-exposed MNL. Monocytemacropage production of membrane-associated IL- I may require pysical contact wit lympocytes as well as accessory inducing signals from lympocytes (23, 37). Te data suggest tat te early abrogation of cell-cell contact also results in diminised soluble IL- 113 production. Furter studies would be required to establis a role for IL-I inibitors specifically in tese events. Production of IFN-'y by T-lympocytes requires monocyte-macropage and lympocyte collaboration (38), but does not require cell proliferation (31). IFN-'y production as been sown to augment te expression of ICAM- 1 on uman memory T lympocytes in an autocrine fasion (32). Tis implies tat ICAM- 1 expression and IFN--y production are associated wit activation after recallenge wit a previously experienced antigen. In vitro exposure of MNL to influenza virus resulted in a prompt, substantial production ofifn-y. In contrast, IFN-,y production by RSV-exposed MNL was delayed, and severalfold lower in magnitude (Fig. 4). Noneteless, IFN-'y production is a measure of immune recognition (24), and its production to any extent supports te concept of preexisting RSV-specific immunity. Terefore, delayed and/or suppressed IFN-'y production may represent anoter in vitro correlate of an abrogated RSV-specific anamnestic response. On te basis oftese data and previous studies (17-19), it is possible to speculate on te sequence of events contributing to a diminised or delayed RSV-specific anamnestic response. Exposure to RSV induces bot IL- 1 inibitor activity and produces suppression of ICAM- I/LFA- I expression. Te pysiologic manifestation of tis suppression, diminised leukocyte clustering, results in limited antigen presentation and te effective reduction or absence of costimulatory signals needed for te clonal expansion of RSV-specific lympocytes. Inibition of IL-l activity affecting lympocytes results in decreased expression of interleukin-2 receptors and transferrin receptors, and a block to lympocyte proliferation (19, 39). Tus, tese studies suggest tat one possible mecanism for te very early inibition of an RSV-specific response is suppression of ICAM-I/LFA- 1 expression and cell clustering. Suc effects would explain, at least in part, te recurrence of RSV in individuals wo sould be immune. Exposure to RSV results in te abrogation ofbot proliferative and nonproliferative RSV-specific responses by uman MNL tat involve collaboration and would be critical for te induction of anamnestic defenses. Te repeated callenge does not result in an expected limitation of virus replication, to levels less tan would produce clinical illness, early after recurrent callenge. Suc observations may also be relevant to te difficult and elusive goal ofdeveloping effective RSV-specific vaccines. Tat is, te events ofsubsequent natural callenge may be a formidable barrier for candidate vaccines tat would rely eavily on MNL recruitment to respiratory defenses. Acknowledaments Tese studies were supported by grants (AI and ) from te National Institute of Allergy and Infectious Diseases. References 1. Werdelin, O., 0. Braendstrup, and E. Pedersen Macropage-lympocyte clusters in te immune response to soluble protein antigens in vitro. I. Roles of lympocytes and macropages in cluster formation. J. Exp. Med. 140: Mock, D. J., F. M. Domurat, N. J. Roberts, Jr., E. E. Wals, M. R. Lict, and P. Keng Macropages are required for influenza virus infection of uman lympocytes. J. Clin. Invest. 79: Rosenstreic, D. L., J. J. Farrar, and S. Dougerty Absolute macropage dependency of T lympocyte activation by mitogens. J. Immunol. 116: Lipsky, P. E., and A. S. Rosental Macropage-lympocyte interaction II. Antigen-mediated pysical interactions between immune guinea pig lymp node lympocytes and syngeneic macropages. J. Exp. Med. 141: Seeger, R. C., and J. J. Oppeneim Synergistic interaction ofmacropages and lympocytes in antigen-induced transformation of lympocytes. J. Exp. Med. 132: Braendstrup, O., 0. Werdelin, E. M. Sevac, and A. S. Rosental Macropage-lympocyte clusters in te immune response to soluble protein antigen in vitro. VII. Genetically restricted and nonrestricted pysical interactions. J. Immunol. 122: Scala, G., and J. J. Oppeneim Antigen presentation by uman monocytes: evidence for stimulant processing and requirement for interleukin 1. J. Immunol. 131: Minceff, M. S., and H. T. Meryman Costimulatory signals necessary for induction of T cell proliferation. Transplantation. 49: Kawakami, K., Y. Yamamoto, K. Kakimoto, and K. Onoue Requirement for delivery of signals by pysical interaction and soluble factors from accessory cells in te induction of receptor-mediated T cell proliferation. J. Immunol. 142: Makgoba, M. W., M. E. Sanders, and S. Saw Te CD2-LFA-3 and LFA-I-ICAM patways: relevance to T-cell recognition. Immunol. Today. 10: Rotlein, R., M. L. Dustin, S. D. Marlin, and T. A. Springer A uman intercellular adesion molecule (ICAM-1) distinct from LFA-1. J. Immunol. 137: Marlin, S. D., and T. A. Springer Purified intercellular adesion molecule-1 (ICAM-1) is a ligand for lympocyte function-associated antigen-1 (LFA-1). Cell. 51: Dustin, M. L., R. Rotlein, A. K. Ban, C. A. Dinarello, and T. A. Springer Induction by ILl and interferon-gamma: tissue distribution, biocemistry, and function of a natural aderence molecule (ICAM-l). J. Immunol. 137: Miller, L. J., R. Scwarting, and T. A. Springer Regulated expression of te MAC- I, LFA- 1, p 150,95 glycoprotein family during leukocyte differentiation. J. Immunol. 137: Dustin, M. L., and T. A. Springer T-cell receptor cross-linking transiently stimulates adesiveness troug LFA-1. Nature (Lond.). 341: Wacoltz, M. C., S. S. Patel, and P. E. Lipsky Leukocyte functionassociated antigen 1 is an activation molecule for uman T cells. J. Exp. Med. 170: McCarty, D. O., F. M. Domurant, J. E. Nicols, and N. J. Roberts, Jr Interleukin- I inibitor production by uman mononuclear leukocytes and 510 A. R. Salkind, J. E. Nicols, and N. J. Roberts, Jr.

7 leukocyte subpopulations exposed to respiratory syncytial virus: analysis and comparison wit te response to influenza virus. J. Leukocyte Biol. 46: Roberts, N. J., Jr., A. H. Prill, and T. N. Mann Interleukin 1 and interleukin 1 inibitor production by uman macropages exposed to influenza virus or respiratory syncytial virus. Respiratory syncytial virus is a potent inducer of inibitory activity. J. Exp. Med. 163: Salkind, A. R., D. 0. McCarty, J. E. Nicols, F. M. Domurat, E. E. Wals, and N. J. Roberts, Jr Interleukin-I inibitor activity induced by respiratory syncytial virus: Abrogation of virus-specific and alternate uman lympocyte proliferative responses. J. Infect. Dis. 163: Kravetz, H. M., V. Knigt, R. M. Canock, J. A. Morris, K. M. Jonson, D. Riflind, and J. P. Utz Respiratory syncytial virus. III. Production of illness and clinical observation in adult volunteers. JAMA (J. Am. Med. Assoc.). 176: Jonson, K. M., R. M. Canock, D. Rifkind, H. M. Kravetz, and V. Knigt Respiratory syncytial virus. IV. Correlation ofvirus sedding, serologic response, and illness in adult volunteers. JAMA (J. Am. Med. Assoc.). 176: Beem, M Repeated infections wit respiratory syncytial virus. J. Immunol. 98: Weaver, C. T., and E. R. Unanue T cell induction of membrane IL 1 on macropages. J. Immunol. 137: Epstein, L. B., M. J. Cline, and T. C. Merigan PPD-stimulated interferon: in vitro macropage-lympocyte interaction in te production of a mediator of cellular immunity. Cell. Immunol. 2: Conmaitree, T., N. J. Roberts, Jr., R. G. Douglas, Jr., C. B. Hall, and R. L. Simons Interferon production by uman mononuclear leukocytes: differences between respiratory syncytial virus and influenza virus. Infect. Immun. 32: Boyum, A A one-stage procedure for isolation ofgranulocytes and lympocytes from uman blood. Scand. J. Clin. Lab. Invest. 21 (Suppl): Domurat, F., N. J. Roberts, Jr., E. E. Wals, and R. Dagan Respiratory syncytial virus infection of uman mononuclear leukocytes in vitro and in vivo. J. Infect. Dis. 152: Roberts, N. J., Jr., and P. K Horan Expression of viral antigens after infection of uman lympocytes, monocytes, and macropages wit influenza virus. J. Infect. Dis. 151: Auron, P. E., S. J. C. Warner, A. C. Webb, J. G. Cannon, H. A. Berneim, K. J. P. W. McAdam, L. J. Rosenwasser, G. LoPreste, S. F. Mucci, and C. A. Dinarello Studies on te molecular nature of uman interleukin 1. J. Immunol. 138: Desroces, C. V., C. Andreoni, and D. Rigal Differential expression of te LFA-I molecule on te uman periperal blood mononuclear cell subpopulations. Immunol. Lett. 24: Kircner, H., R. Zawatzky, H. Engler, V. Scirrmacer, H. Becker, and P. von Wussow Production of interferon in te murine mixed lympocyte culture. II. Interferon production is a T cell dependent function, independent of proliferation. Eur. J. Immunol. 9: Buckle, A. M., and N. Hogg Human memory T cells express intercellular adesion molecule-i wic can be increased by interleukin-2 and interferon-gamma. Eur. J. Immunol. 20: Dougerty, G. J., S. Murdoc, and N. Hogg Te function ofuman intercellular adesion molecule-i (ICAM-1) in te generation of an immune response. Eur. J. Immunol. 18: Roberts, N. J., Jr., andj. Nicols Regulation oflympocyteproliferation after influenza virus infection of uman mononuclear leukocytes. J. Med. Virol. 27: Brownson, J. M., B. W. J. Maey, and B. L. Hazelman Interaction of influenza A virus wit uman periperal blood lympocytes. Infect. Immun. 25: Williams, J. M., B. J. Ransil, H. M. Sapiro, and T. B. Strom Accesory cell requirement for activation antigen expression and cell cycle progression by uman T lympocytes. J. Immunol. 133: Couturier, C., N. Haeffner-Cavaillon, L. Weiss, E. Fiscer, and M. D. Kazatckine Induction of cell-associated interleukin 1 troug stimulation of te adesion-promoting proteins LFA-I (CDl la/cd18) and CR3 (CD1 lb/cd18) of uman monocytes. Eur. J. Immunol. 20: Epstein, L. B., M. J. Cline, and T. C. Merigan Te interaction of uman macropages and lympocytes in te pytoemagglutinin-stimulated production of interferon. J. Clin. Invest. 50: Neckers, L. M., and J. Cossman Transferrin receptor induction in mitogen-stimulated uman T lympocytes is required for DNA syntesis and cell division and is regulated by interleukin 2. Proc. Natl. Acad. Sci. USA. 80: Adesion Molecules and Respiratory Syncytial Virus 511