those obtained from tissues or intact animals. spleen tissue cultures were exposed to LPS at 230C, an early product produced after 24 h of

Transcription

1 INFECTION AND IMMUNITY, Jan. 1977, P Copyright 1977 American Society for Microbiology Vol. 15, No. 1 Printed in U.S.A. Cellular Origin of Interferon Induced by Bacterial Lipopolysaccharide NOBUTOSHI MAEHARA AND MONTO HO* Department of Microbiology and Department of Medicine, School of Medicine, University of Pittsburgh, Graduate School ofpublic Health, Pittsburgh, Pennsylvania Received for publication 21 June 1976 Bacterial lipopolysaccharide (LPS) induces interferons with different properties in mouse macrophages and B lymphocytes. Macrophage interferon is labile at 56 C and is neutralized by anti-mouse fibroblast interferon at a dilution of 1:6,142. B cell interferon is more heat stable and is neutralized by the same antiserum only at a dilution of 1:276. Serum obtained early (1 h) after an intravenous injection of 100,g of LPS resembled macrophage interferon, whereas serum obtained at later times resembled more and more B cell interferon. The diverse cellular origin of LPS-induced interferon may explain the broad hyporesponsiveness produced by LPS in animals. Induction of interferon by lipopolysaccharides (LPS) is similar to induction by other substances in that both messenger ribonucleic acid and protein syntheses are required (10). LPS is still in many ways a unique inducer as compared with more potent substances such as viruses or polyribonucleotides. It is not reactive in ordinary fibroblast or epithelial cell cultures, but it is a B cell mitogen (6) that might induce interferon in the same manner as other mitogens active against lymphocytes such as phytohemagglutinin (21). The study of LPS induction may provide insight into the mechanism of immune induction of interferon (7). Although numerous tissues have been found to produce interferon after LPS is injected in animals, only spleen and thymus tissue cultures are inducible in vitro (9, 10). Which cell is responsible for LPS-induced interferon? The only consistently inducible cell type reported to date is the macrophage, usually obtained from the peritoneal cavity of mice or rabbits (2, 17). Does this mean that macrophages are the sole source of LPS-induced interferon? Probably an indeterminate number of lymphocytes contaminate most of the macrophage preparations used. Epstein et al. (4) pointed out that macrophages must be present for either T or B lymphocytes to produce interferon after stimulation with mitogens, but the producer cells are lymphocytes and not macrophages. One may, therefore, ask whether interferon induced by LPS in macrophage suspension originates from contaminating lymphocytes rather than from the macrophages per se. One approach to this problem is to determine interferon production in purified macrophages or in T or B lymphocytes and to compare the products with those obtained from tissues or intact animals. Previously, Ho et al. (8) showed that when spleen tissue cultures were exposed to LPS at 230C, an early product produced after 24 h of incubation was rapidly inactivated at 56CC, as was LPS-induced macrophage interferon. A later product formed after 24 to 48 h of incubation was more stable a 560C. We suggested that the early interferon represented macrophage interferon, whereas the late-appearing material might represent B cell interferon. To support this hypothesis, direct induction of interferon in B cells by LPS remained to be shown and is reported in this paper. MATERIALS AND METHODS Mice. Five- to eight-week-old, inbred C56 BL/10 male mice (Jackson Laboratories, Jackson, Maine) were used for all purposes except for the preparation of mouse embryo cell cultures, for which pregnant, outbred albino CD-1 mice were used (Charles River Breeding Laboratories, Inc., Wilmington, Mass.). LPS. LPS was prepared by the Boivin method from Escherichia coli 0127 B8 (Difco Laboratories, Detroit,Mich.). It was dissolved in phosphatebuffered saline (ph 7.2) at 10 mg/ml. Media. Unless otherwise specified, "medium" denotes Eagle minimum essential medium in Hanks balanced salt solution containing penicillin (100 U/ ml), streptomycin (100,ug/ml), and 0.08% NaHCO3. "Complete medium" contained, in addition, 5% fetal calf serum. Medium 199 (Grand Island Biological Co., Grand Island, N.Y.) contained the same additives as did complete medium, except that medium 199 substituted for Eagle minimum essential medium and newborn calf serum was used instead of fetal calf serum ALS. Anti-mouse lymphocyte serum (ALS) was prepared in rabbits by the method of Davis et al. (3) 78

2 VOL. 15, 1977 CELLULAR ORIGIN OF LPS-INDUCED INTERFERON 79 and purchased from Microbiological Associates, Inc., Bethesda, Md. Anti-mouse fibroblast interferon antiserum. Anti-mouse fibroblast interferon antiserum was prepared by Kurt Paucker and supplied by the National Institutes of Health (NIH) through G. J. Galasso. It was produced by immunizing rabbits against Newcastle disease virus-induced L cell interferon. Each vial contained 1 ml of lyophilized globulin which, when diluted 1:4,000, neutralized 10 standard reference units of mouse interferon (code G , National Institute of Allergy and Infectious Diseases, Bethesda, Md.) Cell culture. Primary mouse embryo cell cultures from eviscerated embryos were grown in 5-liter Povitsky bottles in medium 199. After 2 days, subcultures were prepared by inoculating 0.1 ml containing 2 x 104 cells in each well of a MicroTest II plate (Falcon) for interferon assay and incubated at 370C in humidified 5% CO2. Glass wool column. A 20-ml plastic syringe was packed to the 8-ml mark with glass wool (fine type, Fisher Scientific Co., Pittsburgh, Pa.), autoclaved, and washed before use with 100 ml of phosphatebuffered saline and 50 ml of complete medium. Nylon wool column. Nylon wool (LP-1 Leukopak, Fenwall Laboratories, Morton Grove, Ill.) was washed with double-distilled water and dried at 370C for several days before use. A 10-ml plastic syringe (Tomac, American Hospital Supply Division of AHSC, Evanston, Ill.) was packed to the 6-ml mark with 0.6 g of nylon wool, sterilized by autoclaving and washed with 50 ml of phosphatebuffered saline followed by 50 ml of medium. Before use, the columns were sealed with Parafilm (American Can Co., Neenah, Wis.), kept at 370C at least 1 h, and washed with 5 ml of warm (370C) medium. Spleen tissue and cell cultures. Mice were killed under ether anesthesia and the spleen was removed. For tissue culture, one spleen was cut in half and incubated in a 25-ml Erlenmeyer flask as described below. For spleen cell cultures, three or four mouse spleens were teased with sterile forceps, filtered through stainless-steel mesh (size 100), and washed two times by adding 10 ml of complete medium and centrifuging at 1,000 rpm for 10 min at 40C. Preparation of macrophages. Mice were exsanguinated by cardiac puncture. A total of 5 ml of medium was injected intraperitoneally and the abdomen was massaged several times. The peritoneal cavity was opened and fluid was aspirated with a no. 19 needle and syringe. The suspension was centrifuged at 1,000 rpm for 5 min and then washed twice with 5 ml of medium. The cells were counted and suspended to a concentration of 2 x 106/ml in medium supplemented with 20% fetal calf serum. A total of 5 ml was placed in a plastic petri dish (60 by 15 mm; Falcon) for 1 h at 370C to permit attachment. The attached cells were washed five times with 5 ml of medium, detached by gentle scraping with a rubber stopper, and resuspended at a concentration of 1 x 106/ml in complete medium. A 1-ml amount was delivered to a stoppered tube for induction of interferon. Separation of T and B lymphocytes. T and B lymphocytes were separated by filtration through glass wool and nylon wool columns (11, 18). A total of 1.5 x 108 spleen cells in 2 ml obtained as described above were poured onto the glass wool column at 230C. This was followed immediately by 50 ml of complete medium. The eluate, devoid of macrophages and dead cells, was centrifuged at 1,000 rpm for 10 min. Cells obtained were resuspended in 2 ml of medium that was loaded onto the nylon wool column. This was followed by 1 ml of warm medium.the column was sealed with Parafilm and incubated at 370C for 45 min. Effluent cells (T cells) were collected by washing columns slowly with 25 ml of warm medium. For elution of nylon-adherent B cells, the columns were rapidly washed with 100 ml of warm medium. The wool was pressed and squeezed several times with stainless-steel forceps, and the cells were collected by washing with 8 ml of warm medium. This operation, including squeezing with forceps, was repeated once. Cells were centrifuged at 1,000 rpm for 10 min at 4 C and resuspended in medium. The number of T cells obtained per column varied from 21 x 106to31 x 106 for T cells and 44 x 106to56 x 106 for B cells. Cytotoxicity test of ALS. A 0.1-ml amount of 1 x 107 cells/ml of lymphocytes or 0.1 ml of 106 macrophages/ml was mixed with 0.1 ml of serial twofold dilutions of ALS, ranging from 1:20 to 1:5,120, to which was added 0.1 ml of a 1:3 dilution of guinea pig serum (complement). All dilutions were made in complete medium. The mixture was shaken and incubated in a 370C water bath for 60 min. After incubation, the tube was put on an ice bath, 0.2 ml of trypan blue solution was added, and viable and dead cells were counted under a microscope. Induction of interferon. To induce interferon in spleen tissue cultures, two slices of tissue were placed in 4 ml of complete medium containing 100,ug of LPS and incubated in a Dubnoff metabolic shaking incubator at 230C and 60 oscillations/min for 24 h. The fluid was then harvested and assayed for interferon. For late interferon production, the early harvest (0 to 24 h) was decanted, and the tissue was resuspended in 4 ml of medium and incubated for another 24 h (24 to 48 h). To induce interferon in cell cultures, 1 ml containing 107 spleen cells or lymphocytes or 106 freshly planted peritoneal macrophages suspended in complete medium was added to 1 ml of medium containing 100,ug of endotoxin. After incubation for 24 h, the cultures were centrifuged at 2,000 rpm for 20 min and the supernatant was stored at -70 C for titration for interferon. Macrophage cultures were always incubated at 230C. To obtain serum interferon, five mice were each injected intravenously with 100,ug of LPS. After stated intervals, blood was obtained from the heart of each mouse and pooled, and the separated serum was stored at -70 C. Interferon assay. Three-day-old subcultures of primary mouse embryonic cells in a MicroTest I plate were used according to the semi-micro, dyebinding assay method of Armstrong (1). Triplicate wells were exposed overnight at 370C to 0.1 ml of twofold dilutions of preparations of interferon to be tested, challenged with 5 x 104 mean tissue culture

3 80 MAEHARA AND HO infective doses of vesicular stomatitis virus in 0.1 ml, and stained with gentian violet for detection of cytopathic effect. Titers expressed in 50% end point dilutions were standardized with the NIH mouse interferon standard. Titration of antigenicity of mouse interferon. About 10 U of an interferon preparation to be tested was mixed with an equal volume of serial twofold dilutions of anti-interferon serum. A series of tubes was incubated at 370C for 60 min, and 0.1-ml portions of each mixture were placed in six replicate wells of a microtray culture. After overnight incubation, the fluids were decanted and each well was challenged with vesicular stomatitis virus. The reciprocal of the dilution of antiserum that permitted development of 50% of the cytopathic effect was considered the titer of the antiserum against a specific interferon preparation. The amount of interferon used was titrated simultaneously, and the dilution end point was adjusted to reflect a titer against 10 U. It was found in separate experiments that the neutralizing power of this antiserum varied proportionately with the concentration of antiserum and inversely with the concentration of interferon. RESULTS LPS-induced interferon produced by macrophages and spleen cells. Peritoneal macrophages respond consistently to LPS by producing modest amounts of interferon. The macrophages must be freshly planted and are quite refractory 4 h after seeding (20). When macrophages were incubated at various temperatures varying from 23 to 370C, interferon was not produced at 370C, but was at lower temperatures (Table 1). In contrast, the temperature optimum of interferon production by spleen cell cultures within the first 24 h was 37CC (Table 1). Late interferon produced by spleen cells between 24 and 48 h of incubation was only demonstrable at 23 and The kinetics of interferon production by macrophage and spleen cell cultures were studied TABLE 1. Interferon production at different temperatures after exposure to LPSa Temp By peritoneal mac- By spleen cells at (h): (OC) rophages at 0-24 h < <2 37 <4 33 <2 a Tube cultures of macrophages or spleen cells were exposed to 100 jig of LPS, incubated at the indicated temperature. To obtain results for production at 24 to 48 h, the cultures were first incubated for 24 h after exposure to LPS, and they were then washed, replenished with medium, and incubated for 24 h more before harvest. at 23 and 370C. Macrophages at 230C produced interferon rapidly in 4 to 12 h with no further production, whereas spleen cells at 230C did not begin producing until after 12 h of incubation. At 370C, spleen cells produced interferon more rapidly (Fig. 1). These data show that the temperature optimum for the production of LPSinduced interferon is different in macrophages than in spleen cells. Production of interferon by purified B and T cells. Portions of 107 B or T cells purified by passage through a nylon wool column were exposed to 100 ug of LPS in 1 ml. The cultures were incubated at 23 or 370C (Table 2). We were unable to detect interferon production by T lymphocytes exposed to LPS. B cells exposed to LPS produced significant but small amounts of interferon between 24 and 48 h at 230C and between 0 and 24 h when incubated at The combination of T and B lymphocytes or macrophages did not produce more than B lymphocytes alone (data not shown). Effect of ALS on macrophages. Macrophages obtained from a mouse peritoneal cavity and attached to a plastic surface appeared to be about 90% pure as judged by light microscopy. To examine the possibility that the presence of lymphocytes was necessary for interferon production, ALS was used. The cytotoxic activity of ALS is shown in Fig. 2. At a final dilution of 1:480 ALS, more than 90%o of a B or T cell suspension was destroyed, whereas 95% of the macrophages remained viable. The effect of varying dilutions of ALS on LPS-induced interferon production in macrophages is also shown in Fig. 2. There was no appreciable difference in the amount produced in macrophage cultures exposed to any dilution of ALS tested, including 1:240, when more than 95% of a test suspension oft and B lymphocytes was destroyed. These results suggest that neitle Cell I, INFECT. IMMUN. 4 5 I 24 4 n INCUBATION TIME k hrs) FIG. 1. Kinetics of LPS-induced interferon production by macrophages and spleen cells at 23 and 37 C. Each point represents a separate cell culture induced by 100 pg of LPS. It represents the total interferon accumulation from the time ofaddition of LPS to the time indicated.

4 VOL. 15, 1977 CELLULAR ORIGIN OF LPS-INDUCED INTERFERON 81 TABLE 2. Interferon production by lymphocytes induced with LPS Temp Incubation Interferon titer" (OC) (h) B cells with LPS T cells with LPS B cells without LPS <4, <2, <2 <4, <2, <2 <4, <2, < , 6, 10 <4, <2, <2 <4, <2, < , 14 <4, <2 <4, < <4, <2 <4, <2 <4, <2 a Each number represents a separate experiment. The interferon produced by B cells induced with LPS was compared with that produced without LPS according to the Wilcoxon rank sum test: (i) when the experimental and control groups were compared, P < 0.05; (ii) when the titers obtained from 24 to 48 h at 23"C and from 0 to 24 hours at 37"C were compared (rows 2 and 3), P < For computation, titers <2 or <4 were assumed to be 2 or O0 9W.0 W OF A L8 DILUTION FIG. 2. Cytotoxicity of ALS on B and T lymphocytes and macrophages and its effect on LPS-induced interferon production in macrophages. ther T nor B lymphocytes are essential for interferon production in macrophage suspensions. Heat and acid stability of LPS-induced interferons. Interferon preparations induced by LPS were obtained from macrophages incubated at 230C and purified B cells incubated at Serum interferons were obtained from mice 2 and 5 h after the inoculation of endotoxin as described above. All four preparations were tested for stability at 56"C (Fig. 3). As reported previously (8), macrophage interferon is heat labile whereas, in contrast, B cell interferon is more heat stable. The serum interferon also varied in its stability; i.e., the early material was more heat labile than the later material (5 h). Thus, in terms of heat stability, early serum from an intact animal resembled macrophage interferon in being labile, whereas the later product was more stable and resembled B cell interferon. Previously (8), a similar variation was noted in activation curves of early (O to 24 h) and late (24 to 48 h) interferon from spleen tissue cultures incubated at 230C. Representative results of experiments testing the stability of interferon preparations at ph 2 are presented in Table 3. None of the interfer- FIG. 3. Inactivation at 56"C ofpurified B, macrophage, and serum interferons induced by LPS. ons was completely destroyed at ph 2 (400) in 24 h. Antigenic characteristics of different LPSinduced interferon. The effect of antiserum against L cell interferon was tested against 10 U of interferon derived from macrophages, B cells, and serum collected at three intervals from mice injected with LPS. The antibody titers of anti-l cell interferon antiserum against interferons from different sources were as follows: macrophage, 6,142; B cells, 276; spleen (at 0 to 24 h and 24 to 48 h of incubation after exposure to LPS), 2,068 and 1,225, respectively; serum (collected at 1, 2, and 5 h after injecting LPS into mice), 4,245, 2,058, and 692, respectively. It is apparent that the highest titer was obtained against macrophage, early spleen, and early serum interferon. The lowest titers were obtained against B cell and later serum interferon. Titers against early spleen and early serum products, although high, were not as high as against macrophage interferon, suggesting that the early products may already be mixed. Similarly, the titers against the late spleen and late serum products were not as low as the titer against B cell interferon. A summary of the characteristics of LPSinduced mouse interferon is presented in Table

5 82 * MAEHARA AND HO 4. The macrophage interferon is heat labile, acid stable, and similar to fibroblast interferon in antigenicity. B cell interferon is different from fibroblast interferon in antigenicity and is more heat stable. Interferon obtained from early spleen cultures and early serum samples from mice injected with LPS has properties similar to macrophage interferon. A different type of interferon, more stable at 5600 but less reactive with anti-fibroblast interferon, is obtained from late spleen harvests and late sera. DISCUSSION Interferons produced in response to bacterial LPS are heterogeneous. Sera obtained from rabbits injected with LPS have a low-molecular-weight (46,000) as well as a high-molecularweight (>100,000) type(12). LPS-induced interferon is also more labile at 5600 and at ph 2 as compared with other interferons, but is still heterogeneous (13). The high-molecular-weight (>100,000) LPS-induced interferon is even more labile than the low-molecular-weight (46,000) product. Salvin et al. (16) reported that interferon induced by old tuberculin in BCG-infected mice, called by them "type II interferon," was completely destroyed at ph 2 in 24 h, whereas that induced by virus or bacterial LPS was not. Similar "immune" interferons induced in human lymphocytes by ALS (5), nonspecific mitogens (4), and specific antigens in sensitized lymphocytes (19) are also more labile at ph 2 and 56 C than virus-induced human interfer- TABLE 3. Stability of LPS-induced interferon at ph 2 INFECT. IMMUN. ons. Immune interferon produced by lymphocytes is also different antigenically from leukocyte (lymphocyte?) interferon induced by a virus (19). These data do not clearly indicate whether interferons are different because they arise from different types of cells or whether one cell type can produce different interferons in response to different modes of induction. Our approach to this problem was to study LPS-induced interferon in purified cells. Any work with the LPS-interferon system is usually beset by low yields. But our results suggest that the cell source influences the properties of the interferon produced. LPS-induced mouse macrophage interferon was heat labile and of similar antigenicity to fibroblast interferon. The early interferons identified in spleen cultures and animals injected with LPS have stability and antigenicity properties similar to macrophage interferon, and we presume that they are identical to it. The late products were more heat stable and not well neutralized by antiserum against fibroblast interferon. On the basis that (i) purified B cells exposed to LPS produced interferon later than macrophages and (ii) B cell interferon was less well neutralized by anti-fibroblast interferon and more heat stable than macrophage interferon, we postulate that the late products represent B cell interferon. These results, of course, do not rule out the possibility that the mode of induction is an additional, independent determinant of the properties of interferon. Indeed this may well be the case since none of our interferons induced by LPS is as labile at ph 2 as Salvin type II interferon. Earlier, on the basis of the finding that thymus tissue exposed to LPS produced interferon Source Temp (0C) of incuba- Collection h Initial titer Titer after 24 h Reduction tion (%) Macrophage B cell Spleen Spleen Serum NAa Serum NA a NA, Not applicable. TABLE 4. Characteristics of mouse LPS-induced interferons Source of LPS-induced interferon Stability to heat Stability to acid (ph 2) Antigenicitya Macrophage Labile Stable High B cells Stable Stable Low Early spleen (0-24 h) Labile Stable High Late spleen (24-48 h) Stable Stable Low Early serum (1 h) Labile Stable High Late serum (5 h) Stable Stable Low a Reaction with antiserum against L cell interferon antibody.

6 VOL. 15, 1977 CELLULAR ORIGIN OF LPS-INDUCED INTERFERON 83 (10, 15), we postulated that T cells produce interferon in response to LPS. We have not been able to detect any interferon production in purified T cell cultures exposed to LPS, and we cannot explain the interferon produced in thymus tissue. Possibly, T cells and macrophages interact synergistically to produce interferon, or macrophages alone are responsible, although we found no evidence for this. Unfortunately, the amount produced in thymus culture was inadequate for further characterization. The hypothesis that LPS-induced interferon in animals originates from at least two different cell types explains certain puzzling phenomena. Macrophage interferon is the rapid and most important component of LPS-induced interferon. These cells are thought to be relatively resistant to irradiation and immunosuppression and account for the continued responsiveness after mice are treated with these measures (see reference 7). Additional LPS action on lymphocytes is important in explaining the broad hyporeactivity against other inducers brought about by LPS in animals, particularly rabbits (9). Lymphocytes are thought to be the main cells reacting after exposure of animals to viruses and polynucleotides (see reference 7). A late interferon produced in B cells in response to LPS may be based on the fact that LPS is a B cell mitogen, whose relationship to immune interferon remains to be determined. Studies in which interferons induced in purified lymphocytes by several inducers are compared are in progress. ACKNOWLEDGMENTS This work was supported by Public Health Service grant 5 ROI-AI from the National Institute of Allergy and Infectious Diseases. We thank our colleague, John A. Armstrong, for helpful criticism and discussion. LITERATURE CITED 1. Armstrong, J. A Semi-micro, dye-binding assay for rabbit interferon. Appl. Microbiol. 21: Borecky, L., V. Lackovic, and K. Waschke Enhancing effect of antiendotoxic serum on interferon induction in mouse peritoneal cells by endotoxin. Acta Virol. 12: Davis, R. C., S. R. Cooperband, and J. A. Mannick Preparation and in vitro assay of effective and ineffective antilymphocyte sera. Surgery 66: Epstein, L. B., M. J. Cline, and T. C. Merigan PPD-stimulated interferon: in vitro macrophage-lymphocyte interaction in the production of a mediator of cellular immunity. Cell. Immunol. 2: Falcoff, E., R. Falcoff, L. Catinot, A. De Vomecourt, and J. Sanceau Synthesis of interferon in human lymphocytes stimulated in vitro by antilymphocytic serum. Eur. J. Clin. Biol. Res. 17: Gery, I., J. Kruger, and S. Z. Spiesel Stimulation of B lymphocytes by endotoxin. Reaction of thymusdeprived mice and karyotypic analysis of dividing cells in mice bearing TJT, thymus grafts. J. Immunol. 108: Ho, M., J. A. Armstrong, and M. C. Breinig Interferon. Annu. Rev. Microbiol. 29: Ho, M., M. C. Breinig, and N. 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Production of endotoxin-induced interferon by mouse spleen cells cultured in vitro. Proc. Soc. Exp. Biol. Med. 131: Kojima, Y Sites of interferon production in rabbits induced by bacterial endotoxin. Kitasato Arch. Exp. Med. 43: Salvin, S. B., J. S. Youngner, and W. H. Lederer Migration inhibitory factor and interferon in the circulation of mice with delayed hypersensitivity. Infect. Immun. 7: Smith, T. J., and R. R. Wagner Rabbit macrophage interferons. I. Conditions for biosynthesis by virus-infected and uninfected cells. J. Exp. Med. 125: Trizio, D., and G. Cudkowicz Separation oft and B lymphocytes by nylon-wool columns: evaluation of efficacy by functional assays in vivo. J. Immunol. 113: Valle, M. J., G. W. Jordan, S. Haahr, and T. C. Merigan Characteristics of immune interferon produced by human lymphocyte cultures compared to other human interferons. J. Immunol. 115: Waschke, K., L. Borecky, and V. Lackovic Release of interferon by mouse peritoneal cells in vitro. The requirement of contact with endotoxin and the temperature dependence of release. Acta Virol. 13: Wheelock, E. F Interferon-like virus-inhibitor induced in human leukocytes by phytohemagglutinin. Science 149: