267 1 tif Archives of Virology 67, 267 281 (1981) Archives of Virology © by Springer Verlag 1981 Differences Between Human c¢ (Leukocyte) and ~ (Fibroblast) Interferons Brief Review By TERESA G HAYES.
Archives of Virology Archives of Virology 67, 267 281 (1981) © by Springer-Verlag 1981 Differences Between Human c¢ (Leukocyte) and ~ (Fibroblast) Interferons Brief Review By TERESA G HAYES Department of Microbiology, New York University School of Medicine, New York, U.S.A Accepted November 24, 1980 Introduction H u m a n interferons are divided into three major classes based on antigenic and physieochemieal criteria The major product of peripheral blood leukoeytes induced with Sendai virus is called :¢ interferon (4, 47) Tissue culture foreskin fibroblasts stimulated with polyinosinie acid-polycytidylie acid [poly (I) • poty (C)] synthesize predominantly ~ interferon (49) ¥ interferon is made by lymphoeytes exposed t~) various mitogenic and immune-specific stimuli, including phytohemagglutinin (40, 114), anti-lymphocyte globulins (39), and viral antigens (8, 45, 109) Recent advances in biochemistry and molecular biology have led to the cloning of the structural genes for some ~ and ~ interferon species (24, 78, 79, 106) The present review is a summary of the various known characteristics of the native a and ~ interferon molecules The review will not attempt to evaluate all of the data obtained in the last few months with cloned interferon DNA sequences, as much of that information has not yet been published Also, as y interferon has been less well characterized than the other two interferon types, it will not be included in the comparison Structural Comparison of Human Intederons Antigenic Properties The first reported demonstrations of human interferon production were performed with primary cultures of amnion and kidney cells stimulated with poliovirus (61, 62) and leukemic bone marrow cell cultures inoculated with several myxo- and paramyxoviruses (56) I t was subsequently established that other human cells could synthesize interferon, including embryonic fibroblasts, cultures from the lung and thyroid, transformed cell lines such as HeLa and KB, and peripheral blood leukocytes (15, 23, 43, 95) 19 Arch Vitol 67/4 0304-860S/81/0067/0267/$ 03.00 268 TERESA G HAYES: As information accumulated about interferons from various sources, it became evident that the products of induced cells were not uniform with respect to their biological and physicochemical characteristics LEvY-KoE~IG et al (70) observed that antibodies raised in rabbits against NDV-induced leukocyte interferon neutralized interferon made by leukocytes to a higher degree than interferon produced in foreskin fibroblasts or amnion cells They concluded that "human interferons derived h'om several types of human cells may exhibit differences in antigenic constitution" This finding was corroborated by DucGoI~A~ et al (28), who noted t~hat rabbit antiserum against white blood cell interferon did not neutralize the antivirM activity of human amniotic membrane interferon in monkey cells A series of experiments employing affinity chromatography with interferon or anti-interferon globulins demonstrated that there are indeed antigenic differences among human interferons An affinity column made of sheep antiserum against interferon from leukocytes did not bind a portion of the interferon activity produced by induced fibroblasts, although leukocyte interferon was completely retained by the column (3) In contrast, Sepharose-couplcd rabbit antiserum against leukocyte interferon bound both leukocyte and fibroblast interferons (4) However, an affinity column prepared with antiserum against fibroblast interferon retained only percent of the activity of a leukocyte interferon preparation (4) To explain these findings, BERG et al (4) suggested that a minor component with the specificity of fibroblast interferon was present in preparations of leukocyte interferon This hypothesis was supported by the work of HAVELL et al (48), who demonstrated that antiserum against leukocyte interferon contains two physically separable antibody populations, one of which binds specifically to an affinity column of fibroblast interferon coupled to Sepharose This indicated that there are at least two antigenically distinct forms of interferon The major antigenic species in leukocyte interferon was designated L~ interferon, while the main component in fibroblast interferon was called F interferon (48) The two interferons are presently called g and ~, respectively (18) When antisera monospecifie for ~ and ~ interferons became available, certain interferon preparations were shown to contai~ varying proportions of the two antigenic types of interferon Interferon induced in peripheral blood leukocytes by Sendal virus had primarily the antigenic specificity- of ~ interferon, with ~ interferon making up less than percent of the total (4, 47) In contrast, ~ interferon comprised 10 to 20 percent of the total interferon activity produced by the Namalva line of lymphoblastoid cells after virus stimulation, with the remMning interferon activity of the ~ antigenic type (i9, 53) Foreskin fibroblasts stimulated with poly (I) • poly (C) synthesized almost exclusively ~ interferon (49, 55) Physicochemieal Properties The g and ~ antigenic types of interferons exhibit certain similarities, as well as many differences, in their physicochemical characteristics The molecular weights of both interferons fall in the same general range, close to 20~000 daltons By mdleeular sieving techniques, ~ interferon appeared to be a homogeneous population with a molecular weight of 25,000 26,000 (6, 14, 38, 107) On sodium dodecyl snifate-polyacrylamide gel electrophoresis (SDS-PAGE), however; ~ interferon Differences Between Human e and ~ Interferons 269 migrated as two fairly broad bands with molecular weights of 15,000 18,500 and 19,000 23,000 (7, 53, 88, 107, t11) Purified ~ interferon produced in lymphobtastoid cells was separated by SDS-PAGE into multiple bands with apparent molecular weights of 18,500 and 22,000; peptide mapping indicated the presence of at least five different ~ interferon subspecies (2) Crude fibroblast interferon was found to have a molecular weight of 26,000 b y sucrose density gradient ultracentrifugation (74) Purified ~ interferon migrated as a single sharp peak of molecular weight 20,000 on SDS-PAGE (65, 99) I n cor~trast to ~ interferon, only one type of ~ interferon has been definitely identified as yet However, the ~ interferon produced by FS-4 tissue culture fibroblasts had a different affinity for the ligand Blue Sepharose than the ~ interferon synthesized by lymphoblastoid cells (37) Two distinct m R N A s coding for ~ interferon have been isolated (85) Thus, there m a y be more than one ~ interferon subspecies as well Both ~ and ~ interferons are highly active molecules The specific activity of and ~ interferons purified to near or complete homogeneity is in ~he range of - - × 10 s units per milligram of protein (65, 66, 82, 99, 118) By definition, a single unit of interferon has antiviral activity Thus, with estimated molecular weights of 20,000, the two interferons are each active on cells at concentrations of about i0 -12 ~ I n some highly sensitive cell-virus systems (e.g., GM-258 cells infected with encephMomyocarditis virus), interferon activity is detectable at 10 -I4 M (J VILS~K, unpublished observations) Studies with chemical reagents suggest t h a t intact disulfide bonds are essential to the activity of both ~ and ~ interferons After reduction of disulfide bonds with ~-mereaptoethano], the two interferons lost antiviral activity (10, 76) Blockage of the reduced sulfhydryl groups by carboxymethylation with iodoacetamide resulted in a further loss of activity for both interferons (76, 99) On the other hand, intact sulfhydryl groups are not likely to be necessary for the action of either interferon, as reagents such as p-ehloromercuribenzoate or N-ethylmaleimide t h a t react with free sulfhydryl groups had no effect, on their activities (10, 76, 99) Despite their general similarities in interactions with sulfur group reagents, and ~ interferons m a y differ when interacting with reducing agents in the presence of denaturants I n a boiling t percent SDS solution, ~ interferon was more stable in the presence of percent ~-mercaptoethanol and ~ Urea t h a n in their absence, whereas ~ interferon had the opposite characteristics (89, 111) The two interferons Mso have different patterns of reaction with several proteolytic enzymes Leueine amino peptidase inactivated a partially purified preparation of interferon to a greater extent t h a n a similar preparation of ~ interferon; e interferon was also much more sensitive t h a n ~ interferon to the actions of carboxypeptidase A and chymotrypsin (80) In general, crude ~ interferon is more stable to heat than crude ~ interferon (reviewed in r d 42) Preparations of ~ interferon were more rapidly inactivated at 56 ° C than preparations of e interferon (12) Studies on purified interferons are difficult to interpret, as thermal inactivation of both interferons is closely dependent on protein concentration, which is not always specified in published reports (65, 84.) However, pa,rtiallh- purified ~ interferon retained all of its antiviral activity 19" 270 TERESA G HAYES: after three days at 56 ° C (75), while partially purified ~ interferon test 57 percent of its activity in 24 hours at 37 ° C (84) Thus, it is likely that in the pure form interferon is more sensitive than ~ to the effects of high temperature In addition, ~ interferon appears to be more susceptible to inactivation by mechanical stress than ~ interferon Vigorous stirring of a crude preparation of interferon resulted in about 80 percent loss of activity after five minutes (50) Rotation end-over-end in a stoppered tube (32) or revolving in a rotational viscometer (11) largely inactivated ~ interferon, whereas z interferon was completely stable to the same treatments The effect seemed to be due to shear forces leading to aggregation of protein molecules, rather than to adsorption to surfaces (10, 11) Both ~ and ~ interferons attach to certain immobilized aromatic ligands The two interferons were retained by a column of the dipeptide L-tryptophyl-Ltryptophan covalently linked to agarose Each required ethylene glycol for elution, confirming the hydrophobic nature of the interaction (93) Nonetheless, there is evidence that ~ interferon has a higher intrinsic hydrophobicity than ~ interferon (64) After binding to the immobilized polyaromatic dye Cibacron Blue F G A (Blue Dextran), ~ interferon could be eluted simply by increasing the ionic strength of the elution buffer, while ~ interferon was not released until after the addition of ethylene glycol (64) In addition, ~ but not ~ interferon bound to CHSepharose (63), L-tryptophyl agarose (93), and controlled-pore glass (33) There is conflicting evidence on whether ~ interferon is a glycoprotein Treatment of ~ interferon with glycosidases resulted in a reduction in molecular weight of all or part of the preparation (6, 7) and a changed pattern on isoeleetric focusing (6, 7, 80) Production of ~ interferon was impaired in the presence of the glycosylation inhibitors tunieamyein (13), glucosamine, and 2-deoxy-D-glueose (91) In addition, sodium periodate treatment of ~ interferon was reported to convert all of the interferon to the lower molecular weight form (90) However, RUBr~ST~I~ et al (82) found at best a very small effect of sodium periodate on the 17,500 molecular weight species of ~ interferon There was little or no binding of ~ interferon to Coneanavalin A, a ]ectin with specificity for Dmannose residues (21, 63, ¥ K YIP, personal communication) Nor did a interferon bind to lectins with affinity for fucose, galactose, N-acetyl neuraminic acid, N-acetylgalaetosamine, ~nd N-acety]glueosamine (63) Furthermore, glueosamine and galactosamine could not be detected in a purified preparation of lymphoblastoid interferon (2) Thus, there is no direct evidence for the presence of sugar residues in z¢ interferon, although it cannot be entirely ruled out at present The data on the glycosylation of ~ interferon are more clear-cut Inhibitors of glycosylation decreased the synthesis of biologically active ~ interferon (51) and elicited the appearance of a smaller molecular weight form of the interferon (52) Treatment of ~ interferon with glycosidases reduced charge heterogeneity and removed sialic acid and N-acetylglucosamine (6) ~ interferon was completely bound by immobilized Concanavalin A (20, 21) In addition, after oxidation with periodic acid, purified ~ interferon stained with Fuchsin base, directly confirming the presence of carbohydrate (65) A portion of an acid hydrolyzate of purified interferon migrated coincidently with galactosamine and/or mannosamine standards (99) Thus, it appears quite certain that ~ interferon is a glyeoprotein Differences Between Human ~ and ~ Interferons 271 Biological Comparison of Human Interferons Antiviral Activity on Human Cells In vitro The first indication that human interferons from distinct sources showed differences in their biological activities came from a study by SUTTOZ¢and TYRI~LL {95) Using crude preparations of interferon, they found that interferon produced in amnion cells had antiviral activity in amnion cells but not in cells from the thyroid, embryonic lung, or kidney Subsequently it was realized that ~ and interferons are both able to protect human fibroblasts against virus challenge, but patterns of antiviral activity differ for the two interferons In human embryo fibroblasts, there was a difference in the slopes of the two dose-response curves (30) In foreskin fibroblasts, the slopes of the dose-response curves for leukocyte and fibrobIast interferons were the same but the cells were much less sensitive to leukocyte than t/o fibroblast interferon (25) Similarly, ~ interferon was generally more efficient than ~ at inhibiting the yield of vesicular stomatitis virus in FS-4 foreskin fibroblast cells; development of the antiviral state was more rapid with interferon in these cells (41, 47) As such studies indicate, the difference in activity between ¢~and ~ interferons appears to be a characteristic of the cells employed in the assay system A direct comparison of cell strains showed that ~ and ~ interferons had identical doseresponse curves in U (human amnion) cells, but in FS-4 cells ~ interferon was more efficient than c~ at producing the antiviral state (11 t) When a standard preparation of ~ interferon was assigned an equal antiviral titer in both cell types, the relative titer of ~ interferon was about ten-fold higher in FS-4 cells than in U cells (59) This indicates that human cells can be preferentially sensitive to one or the other interferon type Antiviral Activity on Heterologous Cells In vitro The differential sensitivity to ~ and ~ interferons is even more pronounced in cells from several non-human species Some heterologous cells appear to be more responsive to ~ interferon than to ~ interferon Interferon induced in human foreskin fibroblasts by Newcastle disease virus was reported to be highly active on primary cultures of rabbit kidney cells, while NDV-induccd human leukocyte interferon had lower levels of activity on these cells (25, 26, 69) A parallel situation was found in rat cells Interferon produced in human amniotic membrane cell cultures induced an antiviral state in rat embryonic fibroblasts, whereas white blood cell interferon was inactive except at very high doses (28) In contrast, many heterologous cells are more sensitive to the ~ type of interferon than to the ~ type H u m a n virus-induced leukocyte interferon had a considerably higher antivirat titer on several bovine and porcine cell cultures than did interferon induced in human embryonic fibroblasts (44, 55) Similarly, a series of feline cell lines was reported to be more sensitive to ~ than to ~ interferon (27) I t should be noted that cells from m a n y heterologous species are insensitive to both c~ and ~ interfcrons (reviewed in ref 87) 272 TERESA G HAYES: Non-Antiviral Activities I n vitro Both ~ and ~ interferons are able to produce the non-antiviral activities associated with interferon Leukocyte and fibroblast interferons were equally effective in enhancing the toxic effect of poly ( I ) poly(C) on fibroblasts (22) Both types of interferon increased cytotoxic T cell activity (58, 117) and augmented the activity of natural killer cells (108, 116) When compared directly, ~ interferon from leukocytes or lymphoblastoid cells caused a similar increase in natural killer activity as ~ interferon from fibroblasts (57) However, the two interferons show certain differences in their non-antivirat actions, especially in their ability to inhibit the multiplication of cells HILFEN~A~rS et al (60) observed that the slopes of the dose-response curves for growth inhibition by ~ and ~ interferons in Daudi cells were different, although the amounts of the two interferons needed for 50 percent growth inhibition were close Later, it was shown t h a t c~ interferon inhibited the multiplication of Daudi cells and another lymphoblastoid cell line to a greater extent than ~ interferon (34) Similarly, g interferon was more inhibitory than ~ to the growth of myeloid progenitor cells (110) I n contrast, ~ interferon was more effective than ~ at suppressing the growth of two osteosarcoma lines (34), virus transformed human embryo fibrohlasts (68), and several non-transformed fibroblast strains (97) ~ interferon prolonged the G1 stage in the life cycle of human fetal skin fibroblasts more strongly than did interferon (71) Thus, as firs~ suggested by EINI~oR~ and STI~ANDEg (3~), the capacity of interferon to inhibit growth m a y be tissue specific: c