Quantitative Studies of the Inflammatory Process in Fatal Viral Meningoencephalitis

Quantitative Studies of the Inflammatory Process in Fatal Viral Meningoencephalitis P. C. Doherty, MVSc, PhD The pathogenesis of acute meningoencephalitis induced in adult mice by intravenous inoculation with Semliki Forest virus has been assessed by counting cells in cerebrospinal fluid (CSF). Meningitis was first apparent on day 4 and, by the time that animals were moribund 2 days later, each microliter of CSF contained in excess of 10,000 mononuclear cells. The following conclusions were made concerning this very considerable inflammatory response: a) Complete suppression of cellular infiltration makes no difference to the clinical disease. b) No correlation is apparent between inflammation and levels of circulating antibody. c) Participation of thymus-derived lymphocytes (T cells) is essential for full expression, though not for initiation, of cellular invasion. d) There is evidently no requirement for lymphocytes recently derived from thymus or for any humoral factor secreted by thymus tissue. e) T cells entering the recirculating pool more than 6 weeks or less than about 1 week prior to inoculation of virus are equally effective in promoting inflammation. f) The T cells apparently act directly by enhancing infiltration of other blood-borne mononuclears into the brain and CSF (Am J Pathol 73:607-622, 1973). A PROTECTIVE ROLE for the inflammatory response has been clearly demonstrated in several acute infectious diseases. The marked cellular infiltration occurring in ectromelia 1,2 and listeriosis 3'4 is associated with elimination of the parasite from the liver and sur- vival of the host. Inflammation is implicated in reducing virus titers in the nonfatal meningoencephalitis caused by intracerebral injection of Sindbis virus." In a few conditions, however, the overall effect of the inflammatory process is deleterious. The severe neurologic symptoms following infection with the closely related lymphocytic choriomeningitis (LCM) and Junin viruses result directly from massive invasion of mononuclear cells into choroid plexuses, men- inges and nervous tissue, rather than from virus-induced dysfunction of nerve cells.6-8 The present study is concerned with the acute fatal meningoencephalitis occurring in adult mice injected intravenously with Sem- From the Department of Microbiology, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia. Accepted for publication July 24, 1973. Address reprint requests to Dr. P. C. Doherty, Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. 607 608 DOH ERTY American Journal of Pathology liki Forest virus (SFV). Particular attention has been given to the role of thymus-derived lymphocytes (T cells) in the disease process. Presence of specifically sensitized T cells is known to. be essential for full development of the inflammatory response in both ectromelia 1,2 and acute LCM, 6,7 though the precise role of such lymphocytes has not been defined. Inflammation in virus infections does not, however, invariably depend on activated T cells. Oldstone and Dixon 9,10 have clearly demonstrated that invasion of mononuclear cells into tissues of persistent LCM carrier mice is secondary to dam- age induced by humoral factors, circulating antibody and complement. Various immunosuppressive treatments have been used to modify the response of mice to infection with SFV. Comparisons of the se- verity of inflammation have been facilitated by a newly developed method for counting cells in mouse cerebrospinal fluid (CSF). This has proved an extremely sensitive assay and may have wider appli- cations in the study of inflammatory process. Materials and Methods Mice Adult CBA/J mice were injected with virus when 8 to 14 weeks old, individ- ual experiments being restricted to animals of the same age and sex. Congenitally athymic nude mice (nu/nu) and their phenotypically normal litter mates (nui/+ ) or ( +/+ ) were used when 4 to 8 weeks old. Virus SFV (strain 2563) was originally obtained from the Yale arbovirus research unit and has been passaged a total of 15 times in adult mice and 4 times in infant mice. Stock virus was stored at -70 C as a suspension of infant mouse brain in gelatine saline. A preliminary experiment established 0.2 ml of a 1OA dilution of this frozen material, 103 plaque-forming units (PFU), as the minimal intravenous dose consistently lethal for adult mice. This dose was used in all experiments. Sampling Technics Mice were anesthetized with ether and exsanguinated. Blood for virologic examination was taken into heparinized gelatine saline, samples for cell counting were diluted in white blood cell (WBC) diluting fluid and smears were made for differential WBC counts. CSF was then obtained by a technic developed by Dr. Richard Carp.1' Skin and muscle are reflected from the atlantooccipital region, thus exposing the bright glistening surface of the dura covering the cistema magna. Using a stereo dissecting microscope, a small hole is made in the membranes with the tip of a 26 gauge needle and CSF is aspirated in a 20-RI pipette attached to a syringe. With a little practice 10 to 15 tl of clear CSF can readily be obtained. Samples were Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 609 December 1973 mixed with cold gelatine saline, diluted in WBC diluting fluid and counted in a hemocytometer or smeared in fetal calf serum and stained with Wright's blood stain. The brain and spleen were then removed. In some experiments half of the brain and most of the spleen were taken into cold gelatine saline, homogenized and centrifuged lightly; the supernate stored at -70 C for virus assay. The remaining tissue was fixed in formalin saline, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Where virus titers were not determined, whole organs were fixed for histology or quenched in liquid nitrogen and used to prepare cryostat sections which were stained by an indirect fluorescent technic to demonstrate SFV antigen. Virus Titration Samples were assayed for SFV on Vero cell monolayers under agar. Virus titers are expressed as log,0 PFU per organ or per milliliter of plasma. Serology Neutralizing index (NI) was estimated for samples of plasma, using a con- stant concentration of plasma and tenfold dilutions of virus in the assay sys- tems described above. Serum hemagglutination-inhibiting (HI) antibody levels were determined following extraction with acetone and ether 12 and are expressed as log,0 of the reciprocal. Specific Antisera Rabbit anti-mouse thymocyte serum (ATS) and anti-O serum, prepared by in- jecting AKR mice with CBA thymus cells, were kindly supplied by Dr. R. V. Blanden.4"13 Mice were given 2 subcutaneous doses of 0.2 ml of ATS or normal rabbit serum (NRS) at 48 and 24 hours prior to the inoculation of virus. Treat- ment with ATS profoundly depletes numbers of circulating lymphocytes.14 Anti-0 serum was used to assess numbers of thymus-derived lymphocytes, bearing the 0 alloantigen,15 in the inflammatory exudate. A 10-,d aliquot of CSF was mixed with an equal volume of either anti-O serum or normal AKR mouse serum and incubated for 30 minutes at 37 C. Samples were then diluted in 2 volumes of guinea pig complement and 1 volume of 0.1% trypan blue. Cells were counted and viability determined after a further 30 minutes at 37 C. When cell suspensions of spleen and thymus 4 from normal CBA mice were so processed, at a concentration equivalent to that found in CSF of clinically af- fected animals (1040/Fd), anti-0 serum killed 94.6 + 1.2% of thymocytes and 36.0 ± 2.7% of spleen cells. This is in reasonable accord with the results of Raff and Wortis.15 Thymectomy and Irradiation Mice were thymectomized (AT.) when 8 weeks old and, if optimal elimination of thymus-derived lymphocytes was required,16 lethally irradiated (850 rads from 60Co source) and reconstituted with bone marrow. Unless otherwise stated ATXBM mice were used 2 to 3 weeks after bone marrow treatment. All such mice were given tetracycline hydrochloride in their drinking water. Statistical Methods All results are expressed as mean ± standard error of mean, log10 for groups of 5 or 6 mice. Groups were compared by the Student t test, using FOCAL on a PDP8-I computer. 610 DOHERTY American Journal of Pathology Results Pathogenesis Development of the disease process was studied by killing mice serially throughout the incubation period. Seventy 10-week-old CBA mice were injected intravenously with 10,f PFU of SFV, and 10 were killed each day: 5 were sampled for virus titration and his- topathology and 5 for cell counts, serology and immunofluorescence. Mice were clinically normal until the sixth day after inoculation, when 16 of the 20 remaining had either died during the previous night (4) or showed evidence of severe neurologic dysfunction (12). Infectious virus was present in plasma and spleen at 24 hours after inoculation and could be isolated from plasma until day 2 and from spleen until day 4 (Text-figure 1). Cessation of viremia preceded detection of free antibody; significant (P < 0.05 or less) levels of neutralizing and HI activity were first demonstrated on day 4. Con- centrations of virus in brain rose steadily until onset of symptoms on day 6, and cells containing viral antigen were apparent from day 5, fluorescence being confined to the cytoplasm of nerve cells. Such 44 u 1 TEXT-FIG 1-Pathogenesis of fatal ¢ 0 meningoencephalitis resulting from 4- intravenous injection of 103 PFU of > Semliki Forest virus. All data are ex- E0 3 pressed as mean logio from 5 mice. ac 2- Serum hemagglutination-inhibit- eU z- ing (HI) antibody titers and values < for neutralizing index (NI) are given 0 as reciprocals.

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