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 survival 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 damage 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 severity 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 systems 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 injecting 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 affected 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

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. (White cells: CSF, 8 stars; blood, open squares. Serum an- 7* tibody: HI, solid squares; NI, circles / with dot. Virus: spleen, circles with 6- dot; brain, asterisks; plasma, open ,5' squares). 4 /\ D

D A Y Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 611 December 1973 neurons occurred in foci which were mainly found in the brainstem and cerebral cortex. Virus was isolated from only one CSF sample, taken on day 4, and antibody was not detected in CSF. Lesions of encephalitis were apparent in 2 of the 5 mice from day 4 and in all of those killed on days 5 and 6. Perivascular cuffing and meningitis were the most obvious pathologic changes but were not especially severe at any stage of the disease. Counting cells in CSF proved a much more sensitive assay of the inflammatory process (Text-figure 1). Normal mouse CSF contains from <10 to a maxi- mum of about 50 cells/[l. Numbers found in CSF were rising on day 3, significantly elevated by day 4; by day 6, CSF of clinically affected mice contained in excess of 12,000 cells/Jl. This inflammatory exudate was almost entirely mononuclear in character (Table 1). Significant numbers of neutrophils were not found in CSF at any stage of the disease process. Percentages of neutrophils in blood were elevated on days 5 and 6, but this was probably due to concurrent depletion of circulating mononuclears as numbers of neutrophils per microliter (mean, 102.9 and 1028) were no different from those in uninoculated controls (mean, 102.8). Serum HI antibody and inflammation were first detected on day 4, and fatal neurologic symptoms were seen 2 days later. By this time severe meningitis was apparent and infectious virus could no longer be isolated from plasma and spleen. Examination at 4 and 6 days after inoculation seemed most likely to illustrate the consequences of various treatments intended to modify the inflammatory process. Nonspecific Immunosuppression Mice were lethally irradiated (850 rads) at 24 hours prior to inoculation of virus, or injected intraperitoneally with 150 mg/kg of

Table 1-Percentage Distribution of White Cells in CSF and Blood CSF Blood Day Mononuclears Neutrophils Mononuclears Neutrophils

3* 100.0 - 0.0 0 84.6 - 2.0 15.4 1.8 4 99.0 - 0.1 1.0 0.7 84.0 - 1.1 16.0 1.1 5 99.6 - 0.0 0.4 0.2 70.8 - 2.4t 28.4 2.7t 6 98.4 - 0.1 2.0 0.7 57.6 - 2.3t 42.6 2-2t * Numbers of cells in CSF were too low for accurate counts to be made on days 1 and 2 t Significantly different (P <0.01) from value for previous day 612 DOHERTY American Journal of Pathology cyclophosphamide (Endoxan-Asta®s, Mead Johnson, Australia) on the day following infection. Both of these procedures are known to result in generalized suppression of the host response to infectious agents.5 17'18 Normal mice survived irradiation for at least 12 day and controls dosed with cyclophosphamide did not die. Irradiation was associated with a significant reduction in survival time and increased virus titers in brain and spleen (Table 2) which may, at least in part, reflect the accompanying persistent plasma viremia (mean, 104-7 and 103.4 on days 4 and 6, respectively). Neither serum HI antibody nor lesions of encephalitis were detected at any stage, and there was no evidence of meningitis on day 4. By the time that animals were moribund, however, CSF cell counts were elevated (P < 0.001) above values for irradiated normal mice (mean, 100.7 cells/,ul). Treatment with cyclophosphamide caused no significant change in either susceptibility or in concentrations of virus in brain, though titers in spleen were considerably increased (Table 2). Serum HI antibody was not detected and numbers of inflammatory cells in

Table 2-Effects of Irradiation and Cyclophosphamide Treatment Days after treatment 850 rads None Cyclophosphamide None Mortality No. dying - 15/15 13/14 15/15 15/15 Daystodeath - 6.0 0.3 8.0 0.6* 6.7 0.3 6.9 0.5 Virus titers Brain 4 7.2 0.2 5.7 0.5* 6.8 0.3 6.2 0.3 6 8.2 0.1 7.4 0.2t 7.6 0.5 7.0 0.4 Spleen 4 4.0 0.1 0.8 0.21 2.9 0.5 0.9 0.6* 6 5.5 0.2 0 3.5 0.4 0 HI antibody No. positive 4 0/5 2/5 0/5 3/5 6 0/5 5/5 0/5 5/5 Titer 6 0 1.6 0.1 0 1.6*0.1 Cell counts (log/10dl) WBC 4 1.0 * 0.3 3.4 0.9t 2.3 * 0.1 3.4 * 0.1t 6 0 3.2 0.1 2.5 0.2 2.7 0.2 CSF 4 1.5 * 0.3 2.7 0.1* 1.8 0.2 3.2 * 0.2t 6 2.6 * 0.2 4.6 0.7t 3.8 0.1 4.3 * 0.1t No. with encephalitis 6 0/5 5/5 5/5 5/5 * P < 0.05 t P < 0.01 t P < 0.001 Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 613 December 1973

CSF were lower than those found in untreated animals at both inter- vals examined. The less severe meningitis on day 4 may, however, simply reflect the much smaller numbers of circulating WBC in these mice. Counts of cells in CSF on day 6 were, on average, five times lower than those in controls, but were still in excess of 2,000 cells/4l. Lesions of encephalitis were apparent in all clinically affected mice. The inflammatory response was almost completely suppressed by lethal irradiation, whereas the effect of cyclophosphamide was much less marked. Onset of neurologic symptoms was not, in any obvious way, dependent on invasion of inflammatory cells. Depletion of Thymus-Derived Lymphocytes Mice were injected with virus subsequent to either administra- tion of ATS or to thymectomy, lethal irradiation and reconstitution with bone marrow (ATXBM). Both processes are known 19,20 to profoundly depress the recirculating pool of thymus-derived lymphocytes (T cells). Neither treatment had any significant influence on survival time, concentrations of virus in tissues or elimination of virus from the spleen (Table 3). Otherwise, the effects of these two procedures were somewhat different. Dosing with ATS resulted in almost complete suppression of inflammation. Numbers of cells per microliter of CSF did not differ significantly at any stage from those found in un- infected ATS-treated mice (mean, 101.4). Despite this profound depression of cellular infiltration, serum HI antibody levels were only slightly lower than titers in controls. However in ATXBM mice inflammatory changes were well developed, but antibody formation was minimal (Table 3). The severity of meningitis was not significantly less than that occurring in intact mice on day 4, though counts made on day 6 were five to ten times lower than those from controls. Remarkably similar results were recorded when congentially athymic nude mice were compared with their phenotypically normal litter mates (Table 4). The process of induction of cellular infiltration is, therefore, apparently similar in ATXBM mice, nude mice and controls. However maximal development of the inflammatory response occurs only in intact animals. The Defect in ATXBM Mice Smaller numbers of inflammatory cells are found terminally in CSF of ATXBM mice than in control mice. Is this due solely to re- 614 DOHERTY American Journal of Pathology

Table 3-Consequences of Depleting Thymus-Derived Lymphocytes Treatment Days after treatment ATS NRS ATXBM None Mortality No. dying - 10/10 9/10 10/10 7/10 Days to death - 9.6 0.9 8.3 0.8 7.4 - 0.5 9.1 0.8 Virus titers Brain 4 4.4 0.5 5.7 0.3 6.6 0.2 6.7 0.2 6 7.1 0.6 7.4 0.1 7.7 0.5 7.7 0.1 Spleen 4 1.4 0.6 1.7 0.6 2.4 0.3 2.4 0.7 6 0 0 0 0 HI antibody No. positive 4 4/5 3/5 0/5 3/5 6 5/5 5/5 2/5 5/5 Titer 6 1.3 0.4 2.1 0.1* 0.4 1.4 - 0.2 Cell counts (log1o/.l) WBC 4 3.4 0.5 3.6 0.4 3.4 0.1 3.1 - 0.1 6 3.3 1.0 3.6 0.3* 3.2 0.1 3.2 - 0.1 CSF 4 1.6 0.2 2.4 0.2t 2.4 0.4 3.2 * 0.3 6 2.2 0.7 4.5 O0.lt 3.5 0.3 4.5 - 0.1* No. with encephalitis 6 2/5 5/5 5/5 5/5 * P < 0.05 t P < 0.01 ATS = antithymocyte serum, NRS = normal rabbit serum; ATXBM = adult thymectomy, lethal irradiation and reconstitution with bone marrow. moval of T cells by ATXBM treatment? Other factors that can be invoked are absence of some "hormone" secreted by the thymus,21 or changes induced by lethal irradiation and reconstitution with bone marrow (BM) that are unrelated to T cell depletion. However, thymectomy 6 weeks previously caused no diminution in either severity of meningitis or in antibody synthesis (Table 5). Cellular infiltration is evidently not dependent on any humoral fac-

Table 4-Meningitis in Nude Mice CSF cells (log1o/Al) Day 4* Day 5*

Nude mice (nu/nu) 2.7 = 0.5 3.9 = 0.1 P < 0.001 Nude control (nu/+) or (+/+) 3.4 - 0.4 4.5 = 0.1 * Mice from day 4 were clinically normal, while those from day 5 were moribund. Lesions of encephalitis were found in all mice from day 5. Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 615 December 1973

Table 5-Influence of Thymectomy and Restoration of Thymic Function on Meningitis and the Serum Antibody Response Weeks after CSF cells (1°g,/1l)* No. with HI antibody final Treatment treatment Day 4 Day 6 Day 4 Day 6 AT,BM 4 2.9 0.3 3.8 0.3t 0/5 2/5 AT, 6 2.7 0.2 4.5 0.1 2/5 5/5 BM 4 2.5 0.3 4.5 0.1 1/5 2/5 None 3.0 0.3 4.6 0.1 3/5 5/5 ATZBM 2 - 3.2 0.3 - 0/5 BM 2 - 3.6 0.2 - 1/5 * WBC counts ranged from 3.0 = 0.1 to 3.4 * 0.1, the only significant difference (P < 0.02) being between the ATXBM (4) and BM (4) groups from day 6. Lesions of encephalitis were apparent in all mice killed on day 6. t Significantly different (P < 0.01) from the other 3 groups in this experiment. AT. = adult thymectomy; BM = lethal irradiation and reconstitution with bone marrow. tor 21 secreted by thymus tissue. Also, though nonthymectomized mice that were irradiated and given bone marrow were no more competent than ATXBM mice at 2 weeks after reconstitution, such animals were able to make a full inflammatory response after a further 2 weeks (Table 5). This is in accord with the observations of Doenhoff and Davies 22 that T cells do not reappear in thoracic duct lymph for at least 3 weeks after irradiation. The T cells required for full expression of the inflammatory process may thus have entered the recirculating pool more than 6 weeks or less than about 1 week previously. How do these T cells promote inflammation? Obvious alternatives are that they could act directly in the lesion, or indirectly via helper function 21 to precursors of antibody-forming cells (B cells) in lymphoid tissue. B Cells and Antibody Any process involving B cells could be mediated either by serum antibody or by circulating B cells localizing in the inflammatory lesion.23'24 However classic B cell progeny25 were not present in CSF in significant numbers, as cells containing immunoglobulins could not be demonstrated with fluorescent antisera (rabbit antimouse Ig, Microbiological Associates Inc., Bethesda, Md). Also severity of inflammation was not, in any previous experiment, obviously related to levels of circulating antibody (Tables 2, 3 and 5). Absence of such a correlation was confirmed by intravenous ad- ministration of 0.5 ml of hyperimmune serum (made from the few 616 DOHERTY American Journal of Pathology surviving mice) to ATXBM mice on day 4. No change was observed in numbers of cells per microliter of CSF 2 days later (mean, 10-5; cF 03-4), even though serum HI antibody titers were quite high (mean, 102.0). A similar result was recorded when cyclophosphamide-treated mice were dosed in the same way with serum from clinically affected animals. Numbers of inflammatory cells in CSF on day 6 were not significantly increased (mean, 1038; cF 1036 in those given control serum). Titers of HI antibody were too low to be detected, although levels of neutralizing activity were sufficient to terminate viremia (mean, 103.4 in controls). The Role of T Cells Potentiation of the inflammatory process by T cells apparently does not operate via helper activity to B cells. The alternative, di- rect involvement of T cells, implies presence of 0-bearing lymphocytes at the site of inflammation. Are significant numbers of T cells found in CSF of clinically affected mice? Treatment with anti-l serum indicated that, as would be ex- pected, CSF of moribund ATXBM mice is substantially free of T cells (Table 6). However in clinically affected intact animals the 0 antigen was present on approximately 20% of invading cells. Thus, the presence of thymus-derived lymphocytes alone does not account for the 80 to 90% difference between numbers of inflammatory cells found terminally in CSF of ATXBM and control mice. The T cells apparently amplify the inflammatory process by enhancing infiltration of other blood-borne mononuclears into CSF. Discussion Several points require further consideration: a) The large numbers of inflammatory cells entering brain and CSF have little influence on either the development of neurologic symptoms or on the

Table 6-Effect of Anti-O Serum on Inflammatory Cells in CSF of Mice Given SFV 6 Days Previously Percent of cells killed CSF cells Host treatment (log1o/zl) Normal serum Anti-e serum Difference

None 4.2- 0.1 11.6 - 1.8 29.3 -3.3* 17.6 4.3 None 4.5 - 0.1 13.7 - 2.3 33.0 f 3.2* 19.3 3.0 ATXBM 3.5 0.2 14.1 - 3.3 12.3 = 5.0 * Significantly greater (P < 0.01) than value for normal serum. Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 617 December 1973 survival of the host. b) Although both ATXBM treatment and dosing with ATS cause considerable depletion of T cells, depression of the inflammatory process is much greater in mice given ATS. c) Induc- tion of meningoencephalitis is not readily shown to be an immuno- logically specific process. d) Development of maximal inflammation requires participation of thymus-derived lymphocytes, which amplify the inflammatory response by enhancing infiltration of other blood-borne mononuclears.

Clinical Consequences and the Source of Cells Deletion of the inflammatory process did not obviously modify the course of this almost invariably lethal disease. In other viral en- cephalitides, suppression of cellular invasion generally increases mortality, although in some instances the effect is minimal and time to death is slightly delayed.18 23'26'27 With the exception of LCM and other arenavirus infections,6'8 there is little experimental evidence to support the view that inflammation is paramount in the production of neurologic symptoms.28 Inflammatory cells are presumably recruited from the circula- tion.29 It is most unlikely that significant numbers originate endo- genously within the central nervous system. Even in normal animals, many "microglial" cells are probably derived from blood mono- cytes.30 Cells in nervous tissue would not be particularly accessible to ATS,14 but such treatment resulted in considerable diminution of the inflammatory response both in these experiments and in the brains of rats implanted with skin homografts.31 Also, suppression of inflammation subsequent to lethal irradiation or treatment with cyclophosphamide parallels depletion of blood leukocytes.

Dosing with ATS and ATXBM Mice Cellular infiltration was minimal in animals injected with ATS, although the levels of circulating antibody were almost normal. The reverse applied in ATXBM mice. Inflammation was quite severe, but little antibody was formed. Dosing with ATS affects circulating lymphocytes preferentially: penetration of lymphoid tissue is poor.14 The heterologous antisera used can be detected in blood for as long as 6 days after inoculation 32 and may contain additional antiinflam- matory components which are not immunoglobulins.33 Following ATXBM treatment, however, populations of 0-bearing cells in spleen and lymph nodes are greatly reduced,'5 and numbers of circulating T cells are less than 10% of normal levels.34 Nude mice 618 DOHERTY American Journal of Pathology are completely free of T cells 15,35 and behave much like ATXBM animals. The disease pattern in ATXBM and nude mice should thus be a more valid reflection of depressed T cell function than is that found subsequent to treatment with ATS.

Induction of the Inflammatory Process Initiation of invasion by blood-borne mononuclears was not shown to be dependent on any component of the specific host response. Re- sults from ATXBM and nude mice indicate that sensitized T cells are not essential for "triggering" the inflammatory process, a situation quite different from that found in ectromelia2 and LCM.7 No correlation was apparent between levels of circulating antibody and cellular infiltration. Dosing with cyclophosphamide affects B cells rather than T cells.36'37 Marked inflammatory lesions were seen in mice given cyclophosphamide at a level sufficient to completely sup- press inflammation in LCM 6 and in Sindbis virus encephalitis.5 The important difference is that SFV is, of itself, extremely virulent for mice. The virus kills in the absence of inflammation, whereas infection with Sindbis virus is normally asymptomatic, and LCM is clinically inapparent in immunosuppressed animals. Onset of inflammation at 4 days after intravenous injection of SFV may thus result directly from virus-induced damage to the CNS. Cells infected with viruses are known to release various factors which are chemotactic in vitro for leukocytes. Some, but not all of these substances, operate by activating complement.3840 However, neutrophils are attracted by the preparations examined so far at least as strongly as are macrophages, and neutrophils did not at any stage form a significant component of the cellular exudate in mice infected with SFV.

Maximal Inflammation and T Cells Even in clinically affected animals there does not seem to be a generalized breakdown of the blood-CSF barrier, for neutrophils and red blood cells are largely excluded from CSF. Chemotaxis resulting from viral cytopathic effects may be responsible for the quite marked meningoencephalitis observed in moribund ATxBM mice. Augmentation of this apparently nonspecific background of inflammation requires participation of T cells. A similar situation has been described for granulomas induced by injecting Bordatella pertussis or BCG into the feet of mice.41 Some properties of these T cells are reasonably well established. Vol. 73, No. 3 QUANTITATION OF VIRAL MENINGOENCEPHALITIS 619 December 1973 As in ectromelia,2 the T cells augment the inflammatory process by acting directly in the target organ. In this context, CSF may be con- sidered as a "sink" for products of virus-induced damage to nerve cells.42 The age of T cells involved does not seem to be important: there is no necessity for lymphocytes recently derived from thymus,3 a finding also made in studies of listeriosis.4 The probable mechanism of amplification is that specifically sensitized T cells circulate relatively late in the disease and enter brain and CSF, along with other blood-borne mononuclears, subsequent to virus-induced damage to the central nervous system. Extravas- ated lymphocytes react with viral antigen and release various chem- ical mediators of inflammation 4445 which recruit more circulating mononuclears into lesions. This results in concentrations of leukocytes in CSF of from ten to fifty times those in an equivalent volume of blood.

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Acknowledgments I wish to thank Miss Gail Essery for her enthusiastic technical assistance and Mr. R. Hill for preparing the histologic sections. 622 DOHERTY American Journal of Pathology

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