Production of Chemotactic Factor and Lymphotoxin by Human Leukocytes Stimulated with Herpes Simplex Virus

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1 INFECTION AND IMMUNITY, July 197, p Copyright 197 American Society for Microbiology Vol. 1, No. 1 Printed in U.S.A. Production of Chemotactic Factor and Lymphotoxin by Human Leukocytes Stimulated with Herpes Simplex Virus GARY L. ROSENBERG, RALPH SNYDERMAN, AND ABNER L. NOTKINS Laboratory of Oral Medicine, National Institute of Dental Research, Bethesda, Maryland 21, and Division of Rheumatology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 2776 Received for publication 18 March 197 Peripheral blood leukocytes (PBL) of 1 subjects were tested for their ability to be stimulated by herpes simplex virus antigens as measured by [3H ]thymidine incorporation and lymphokine production (i.e., lymphocyte-derived chemotactic factor and lymphotoxin). The PBL of seronegative subjects failed to respond to viral antigens as measured by stimulation or lymphokine production. In contrast, PBL from seropositive subjects without clinical lesions of herpes labialis were stimulated by herpes simplex virus antigens and produced lymphokines. PBL from seropositive patients with clinical lesions of herpes labialis also produced lymphokines, but [3H ]thymidine incorporation was slightly depressed. These findings suggest that lymphocytes from patients with a recent herpes simplex virus infection may respond less vigorously to in vitro stimulation by herpes antigens, but our study fails to show any basic defect in the responsiveness of the lymphocytes to account for recurrent herpetic episodes. Recurrent herpes simplex virus (HSV) infection is one of the most common viral afflictions of man. Neither the factors responsible for recurrence nor the immunological mechanisms by which the host attempts to control this infection have been clearly elucidated. Since the infection persists and can spread in the presence of high concentrations of neutralizing antibody (11), something more than antibody is needed to stop the infection. Recent experiments suggest that antibody, complement, immune lymphocytes, lymphokines, and macrophages contribute to the defense against this infection (9). The possibility that a deficiency in one or more of these factors might contribute to recurrence has been suggested. The present experiments were initiated to see whether human peripheral blood leukocytes (PBL) could be stimulated by HSV antigens to produce lymphokines and whether there was any difference in the response of PBL from subjects with and without clinical symptoms. MATERIALS AND METHODS Subjects. Subjects were divided into groups on the basis of serum antibody levels to HSV and the presence or absence of active herpes labialis. Group 1 consisted of 13 patients with active herpetic lesions and neutralizing antibody to HSV. Group 2 consisted of 21 subjects who had neutralizing antibody to HSV, but who either had no history of recurrent herpetic lesions or were free of lesions for at least months. Group 3 consisted of 7 subjects with no antibody to 111 HSV and no clinical history of HSV infections. At the time of examination the subjects had no major illness and their age ranged from 12 to 55 years. Viral antigens and neutralizing antibody determinations. The methods employed in this study have been described in detail elsewhere (, 7, 1, 16). HSV (type 1) was prepared and assayed in primary rabbit kidney (PRK) cells grown in the presence of 5% rabbit serum. Virus was partially purified by ultracentrifugation and suspended in Dulbecco's phosphate-buffered saline containing Ca2l and Mg2+ (PBS) at a titer of 1. x 19 plaque-forming units per ml. For in vitro lymphocyte stimulation studies, partially purified virus was inactivated by exposure to ultraviolet light and stored in ampoules at -2 C. Antibody to HSV was quantitated by the plaqueneutralization technique. The titer is expressed as the reciprocal of the highest serum dilution that reduced the number of plaques by 5% as compared to controls (7) Ṡtimulation of PBL Cultures. The preparation and in vitro stimulation of PBL by HSV antigens, with minor modifications, have been described previously (, 1). PBL were obtained from heparinized blood after sedimentation of the erythrocytes with dextran. The cells were washed and suspended at a density of 2. x 16 cells/ml in RPMI 16 medium supplemented with 15% autologous plasma, glutamine, and antibiotics. In a typical experiment, 1. ml of cell suspension was cultured in triplicate with.5 ml of ultraviolet-inactivated HSV. PBS and a cell homogenate from uninfected PRK cultures, prepared by ultracentrifugation as in the case of viral antigen, served as controls. Cultures were set up in screw-cap glass vials and incubated at 37 C for 72 h in a Downloaded from on August 17, 218 by guest

2 112 ROSENBERG, SNYDERMAN, AND NOTKINS humidified atmosphere of 5% CO2 and 95% air. For the final h of culture, 1. yci of [3H]thymidine (specific activity 18 Ci/mmol) was added to each culture vial. Cultures were harvested on membrane filters (Millipore Corp.), and incorporation of [3H]thymidine was determined by liquid scintillation spectrometry (). All values for triplicate cultures were within ±+1% of their mean. Results are expressed as the ratio (stimulation ratio) of the mean counts per minute incorporated in the presence of viral antigen to the mean counts per minute incorporated in the presence of PBS. To show that PBL preparations were capable of being stimulated in vitro, 3 ug of phytohemagglutinin (PHA, batch E316/Q1, Burroughs-Wellcome, Tuckahoe, N.Y.) was added to triplicate cultures. In every case greater than 8-fold stimulation was observed. Production of lymphokines. PBL cultures were established in parallel for collection of leukocyte supernatant fluids for lymphokine assay. In patients with active herpes labialis, lymphokine production was studied 7 to 1 days after onset of recurrence. For lymphotoxin production, 2. x 16 PBL/ml in 6. ml of RPMI 16 medium supplemented with 15% autologous plasma, glutamine, and antibiotics were incubated for 12 h with.3 ml of ultraviolet-inactivated HSV, PHA, PBS, or PRK cell homogenate. Supernatant fluids then were aspirated, clarified by centrifugation at 1,5 x g for 15 min and stored at -2 C until tested for lymphotoxin activity. For quantitation of lymphocyte-derived chemotactic factor production, 3. x 16 PBL/ml in 6 ml of RPMI 16 medium containing.5% autologous plasma were incubated for 72 h with ultraviolet-inactivated HSV, PHA, PBS, or PRK cell homogenate. Supernatant fluids were then aspirated, heated at 56 C for 3 min, clarified by centrifugation, and stored at - 2 C until tested for chemotactic activity. Lymphotoxin assay. Lymphotoxin was assayed by a modification (8) of the method of Granger and Kolb (6). Muse L-cell monolayers in loosely capped, 1- dram glass vials were incubated at 37 C for 8 h with 1 ml of a 1:1 mixture of supernatant fluid from stimulated leukocyte cultures and leucine-free RPMI 16 medium. For the final 2 h of culture,.5 gci of L- [1C ]leucine (specific activity 312 mci/mmol) was added to each culture vial. The amount of L- ["C 1- leucine incorporated into the trichloroacetic acidprecipitated fraction of quadruplicate fibroblast cultures then was determined. Lymphotoxin activity is expressed as the percent inhibition of L-[C]Ileucine incorporation into mouse L-cells incubated with supernatant fluids from PBL cultures that had been exposed to HSV antigens or PHA as compared to incorporation by L-cell monolayers incubated with antigen-reconstituted supernatant fluids from PBL cultures that had been exposed to PBS or PRK cell homogenate. Chemotactic factor assay. Chemotaxis of human mononuclear leukocytes was measured by a modification (19) of Boyden's technique (1). Human mononuclear leukocytes were obtained from heparinized peripheral blood of healthy volunteers by the Ficoll- Hypaque method (2), washed with Gey's balanced salt solution (ph 7.), and placed on one side of a 5.-pm polycarbonate filter (Nucleopore, Wallabs, Inc., San Rafael, Calif.). A standard dilution of leukocyte supernatant fluid then was placed on the other side of the filter and chemotactic activity was quantitated by counting the number of human mononuclear leukocytes that migrated through the filter toward the test substance during a 9-min incubation at 37 C. Assays were performed in triplicate. Leukocytes in 2 microgrid fields (99x) were counted, averaged, and expressed as the number of cells per oil immersion field. The chemotactic index represents the number of human mononuclear leukocytes per oil immersion field that migrated through the filter toward supernatant fluids from PBL cultures that had been exposed to HSV antigens or PHA, divided by the number of cells that migrated through the filter toward supernatant fluids from PBL cultures that had been exposed to PBS or PRK cell homogenate and reconstituted with HSV antigens. Chemotactic factors of known activity (i.e., C5a and lymphocyte-derived chemotactic factor produced by PHA stimulation of normal human leukocytes) were used as positive controls (19). RESULTS I NFECT. I MMUNITY Stimulation of PBL by HSV antigens. The responses of individuals with antibody to HSV but no active lesions (group 2) and individuals with neither antibody to HSV nor a history of HSV infection (group 3) are illustrated in Fig. 1. The stimulation ratio of the seropositive group was (mean + standard error of the mean). The geometric mean neutralizing antibody titer of this group was 1,58. In contrast, subjects without antibody to HSV (neutralization titer <) had a mean stimulation ratio of One of the seven seronegative subjects showed slight stimulation to viral antigens. However, this subject responded to approximately the same degree to PRK cell homogenate, suggesting that he was sensitive not to viral antigens, but to rabbit antigens which might be associated with the tissue culturegrown virus. The response of 13 seropositive subjects with active recurrent herpetic lesions is illustrated in Fig. 2. Each subject was seen within 1 to 3 days after the onset of lesions and bled three times during the course of the study. The stimulation ratio (mean : standard error of the mean) at 1 to 3 days was This did not differ appreciably from the mean stimulation ratio of the seropositive lesion-free subjects (Fig. 1). However, at 7 to 1 days and 28 to 31 days after onset of symptoms, the stimulation ratio was and.5 +.7, respectively. Eleven of 13 patients had a drop in stimulation ratio from the first to the second bleeding, and 12 of 13 showed a decrease in the stimulation ratio from the second to the third bleeding. The fall in stimulation ratio from the initial to the final Downloaded from on August 17, 218 by guest

3 VOL. 1, o 2 I- Z 16 -J 2 12 ~~7- ~ 1 8 CHEMOTACTIC FACTOR AND LYMPHOTOXIN PRODUCTION % ANTIBODY ANTIBODY POSIT VE NEGATIVE FIG. 1. Stimulation by HSV antigens of PBL from subjects with antibody to HSV but no active clinical lesions as compared to seronegative subjects with no history of clinical lesions. Bars represent the mean stimulation ratio ± standard error of the mean o 2 cr z 6 -J E 12 I- cn 8 _ i IO DAYS 28-3 FIG. 2. Stimulation by HSV antigens of PBL from seropositive patients with active recurrent herpes labialis. Days indicate time after onset of lesions. Bars represent the mean stimulation ratio + standard error of the mean. * _ * I ' -T-~~~~~~~~~~~ bleeding was statistically significant, P <.5. Production of lymphokines. The data in Table 1 show that supernatant fluids from PBL of seropositive patients that had been stimulated with HSV antigens contained significant amounts of lymphotoxin activity. PBL of subjects who were lesion free produced approximately the same amount of lymphotoxin as PBL of patients who were suffering from acute herpes labialis. In contrast, PBL of the seronegative group produced little if any lymphotoxin. The one exception, TC, is the same individual whose lymphocytes were stimulated by PRK cell homogenate. That cells of seronegative subjects are capable of producing lymphotoxin was demonstrated by their ability to elaborate the lymphokine in response to PHA. TABLE 1. Lymphotoxin production by peripheral blood leukocytesa Supernatant fluids from PBL stimulated with: Clinical status HSV PHA Inhibi- Inhibition Pb tion (%) (%MY Antibody positive Lesions present DB 21.7 < MC 16.9 <.1 EH 23.3 < EMH 39. < JP 31.1 < MR 2.9 < ES 53. < NS 19.1 <.1 DW 33.6 <.1 Avg Lesions absent AN 15.6 <.1.3 GR 35.8 < JR 3.5 < MR 18.5 <.1 CW 26.2 <.1 RW 57. < Avg Antibody negative WA 2.2 > JC -2.8 > TC 13.8 < GP 2.9 > AS.3 > Avg a Mouse L-cell monolayers were incubated for 8 h with supematant fluid from PBL that had been incubated with HSV, PHA, or PBS. L- [C ]leucine incorporation then was measured. Lymphotoxin activity is represented as percent inhibition of L- [C ]- leucine incorporation by experimental (HSV, PHA) as compared to control supernatant fluids (PBS). Pp values from Student's t test. c All values for percent inhibition by PHA supernatants are significant, P <.1. Downloaded from on August 17, 218 by guest

4 11 ROSENBERG, SNYDERMAN, AND NOTKINS INFECT. IMMUNITY Table 2 shows that supernatant fluids from PBL of seropositive patients that had been exposed to HSV antigens produced significant amounts of lymphocyte-derived chemotactic factor irrespective of whether they had clinical lesions. Except for T.C., all seronegative subjects failed to produce significant amounts of lymphocyte-derived chemotactic factor. Chemotactic factor was readily produced when the PBL of the seronegative subjects were exposed to PHA. DISCUSSION It is well established that patients with immunological deficiencies are more susceptible to certain types of viral infections (5). Moreover, there is evidence that infections such as chronic mucocutaneous candidiasis may be associated with deficiencies in lymphokine production (13, 17, 18). With the development of in vitro assays for studying cellular immunity and measuring TABLE 2. Production of lymphocyte-derived chemotactic factor by HSV-stimulated peripheral blood leukocytes Supernatant fluids from PBL stimulated with: Clinical status HSV PHA Cla P CI P Antibody positive Lesions present DB 3.7 <.5.7 <.5 MC.2 <.5 EH 2.9 < <.2 EMH 9.7 <.1 MR 1.8 <.1 ES 3.6 <.5 NS 3.2 <.1 Avg 5.. Lesions absent AN 3.6 <.5. <.2 GR 19.3 <.1 JR 1. <.5 CW 12.7 <.1 RW 3.3 < <.2 Avg Antibody negative WA 1. >.25.6 <.1 JC 1.2 >.5 3. <.1 TC. < <.1 GP 1. >.5 AS 1.6 > <.1 Avg a Chemotactic index (CI) was calculated as described in Materials and Methods. The CI with C5a and a standard preparation of lymphocyte-derived chemotactic factor were 27.3 and 31.1, respectively. Pp values from Student's t test. biological mediators produced by lymphocytes, it has become possible to evaluate these factors in patients with HSV infections. Our studies show that PBL from patients who are seropositive to HSV can be stimulated in vitro by HSV antigens. A slight decrease in the stimulation ratio was noted during and after recurrences, but because of the small number of patients studied, these observations, although statistically significant (P <.5), need to be confirmed, and the biological significance, if any, should be determined. In addition, our studies showed that exposure of PBL from seropositive patients to viral antigens led to the production of lymphotoxin and lymphocytederived chemotactic factor. Approximately the same amount of lymphotoxin was produced by seropositive subjects with and without lesions, and although it appears that slightly less chemotactic factor was produced by the group with lesions, this difference is not statistically significant (P >.1). Moreover, analysis of the data from individual seropositive patients failed to reveal any correlation between the titer of neutralizing antibody, the degree of lymphocyte stimulation, and the amount of lymphokines produced. Recently, other investigators (12, 22) also showed that PBL from seropositive patients could be stimulated by HSV antigens. No real difference in the degree of PBL stimulation was found when patients with and without active lesions were compared. Wilton et al. (22), however, reported that the production of migration inhibition factor was somewhat depressed in patients with recurrent HSV attacks as compared to controls who did not get recurrent attacks. Since a large percentage of these controls (6%) also did not make migration inhibition factor, it would appear that a deficiency in migration inhibition factor production cannot be the only explanation for recurrences. The ability of PBL from seropositive patients to produce interferon upon exposure to HSV was recently reported by Rasmussen et al. (12). In addition, these investigators observed that the interval between recurrences was considerably shorter in patients whose PBL produced low amounts of interferon when tested at 2 to 6 weeks after the onset of lesions. Since relatively little is still known about the temporal and quantitative relationships between lymphocyte stimulation and lymphokine production in acute viral infections, the biological significance of these observations remains to be determined. Moreover, even if lymphocyte stimulation and lymphokine production are depressed during or after recurrent HSV attacks, this could very Downloaded from on August 17, 218 by guest

5 VOL. 1,197 CHEMOTACTIC FACTOR AND LYMPHOTOXIN PRODUCTION well be the result of the infection (e.g., overstimulation of lymphocytes by continued exposure to viral antigens; removal of specific subpopulations of lymphocytes from peripheral circulation to the site of the lesion) rather than the cause of recurrence. In this connection, lymphocyte stimulation and migration inhibition factor production were highest at 7 to 1 days after primary infection of animals with HSV and influenza virus, but declined markedly over the next 1 days (1, 15, 21). What is evident from our studies and those of Wilton et al. (22) and Rasmussen et al. (12) is that PBL of seropositive patients produce lymphokines upon exposure to HSV antigens. These lymphokines may very well play a critical role in combating the viral infection. Chemotactic factors can attract leukocytes to the site of the infection; migration inhibition factor can activate and keep them there; lymphotoxin can aid in destroying both infected cells and nearby uninfected cells to which the virus would ordinarily spread; and interferon can inhibit viral replication (3). The question of whether there is a deficiency in lymphocyte stimulation and/or lymphokine production in patients with recurrent HSV infections clearly requires further study. If there is a deficiency, it remains to be determined that it is of sufficient magnitude to have biological significance in terms of influencing the frequency and duration of recurrent infections. Alternatively, if a defect in cellular immunity cannot be substantiated, this would suggest that the problem of recurrences lies not in the immune response of the host, but in factors responsible for the activation of HSV in the trigeminal ganglion of the asymptomatic host (2). ACKNOWLEDGMENTS We thank Edward Graykowski, Takashi Fujibayashi, Shirley Geis, Christine Stahl, and Linville Meadows for their assistance in this study. G.L.R. is a recipient of Public Health Service special fellowship 6F3 DE532-1 from the National Institute of Dental Research. R.S. is a Howard Hughes Medical Investigator. This work was supported in part by Public Health Service grant 1ROlDE from the National Institute of Dental Research. LITERATURE CITED Boyden, S. J The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115: Boyum, A Isolation of leukocytes from human blood. Further observations; methylcellulose, dextran and ficoll as erythrocyte-aggregating agents. Scand. J. Clin. Lab. Invest. 21(suppl. 97): David, J. R Lymphocytic mediators and cellular hypersensitivity. N. Engl. J. Med. 288: Elfenbein, G. J., and G. L. 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