Microinjection ofAnti-Interferon Antibodies intoCells DoesNot Inhibit theInduction ofan Antiviral State byInterferon

Affinity-purifie d polyclonal antibodies directed against humanlymphoblastoid interferon (IFN), Escherichia coli-derived humanIFN-a2, ortwosynthetic fragments ofhumanIFN-otL allneutralized theantiviral activity ofhumanaIFNswhenaddedtotheculture mediumofMDBK cells together withIFNs. However, whenthese antibodies weremicroinjected into thecytoplasm orthenucleus ofcells, subsequent treatment ofthecells with IFNsinduced full protection against vesicular stomatitis virus. This suggests that IFNsthemselves neednotact inthecytoplasmic compartment orthenucleus toinduce an antiviral state. Itappears that theinteraction ofinterferons (IFNs) witha small numberofhigh-affinity binding sites present onthe surfaces ofsensitive cells issufficient toactivate cellular genesandtoinduce anantiviral state (1,2).However, itis unknown howthesmall number ofligand-binding receptors transfers signals fromtheplasma membrane tointracellular compartments. Itispossible thatthegeneration ofsuch signals isthesoleproperty ofthereceptor molecules and thattheligand itself hasnootherrolethanactivating receptors byspecifically binding tothem.However, ithas recently beenshownthat receptor-bound IFNsareinternalizedanddegraded bycertain cells (3,6,13)andthat internalization follows aspecific pathway knownasconcentrative adsorptive (receptor-mediated) endocytosis (13). Therefore, itisconceivable that IFNsortheir degradation products aredelivered tothecytoplasm (andperhaps tothe nucleus), wheretheycould directly interact withhostregulatory mechanisms. Suchacytoplasmic site ofaction hasfor instance beendescribed fordiphtheria toxin, whichislikewiseinternalized byreceptor-mediated endocytosis. Ithas beenshownthatintroduction ofantibodies todiphtheria toxin into cells protects these cells against thelethal effect of subsequent exposure tothetoxin (12). Toinvestigate apotential cytoplasmic orintranuclear site ofaction ofIFNs,we microinjected specifically purified polyclonal antibodies toIFNinto cells before treating the cells withIFN.Thefollowing preparations ofantibodies wereusedforthese studies. (i) Sheepserumobtained after immunization withpurified human(Hu)IFN-alymphoblastoid(ly) wasfractionated asdescribed previously (14). Specific immunoglobulin G (IgG) wasobtained byaffinity purification overanAffi-Gel 10columncontaining 5mgof purified recombinant HuIFN-aL2 (agift ofSchering Corp., Bloomfield, N.J.) perml,andthefraction eluting atpH2.6 wascollected. Endpoint titration oftheIFN-neutralizing activity ofthis antibody preparation showedthat 0.05ngof HuIFN-a.(ly) perml(which equals 10antiviral U/ml) was neutralized by0.2ngofantibody perml.Normalsheep IgG hadnoIFN-neutralizing activity. (ii) Rabbit serumobtained after immunization withpurified recombinant Hu IFN-a2 wasfractionated byammoniumsulfate precipitation, passageovera protein A-Sepharose column, andfinally by passage oftheprotein A-binding fraction overtheHuIFN