Parainfluenza infection of aged mice results in autoimmune disease

CLINICAL

IMMUNOLOGY

AND

Parainfluenza

IMMUNOPATHOLOGY

12, 301-315 (1979)

Infection of Aged Mice Results Autoimmune Disease’ MARGUERITE

in

M. B. KAY

Molecular and Clinical Immunology Laboratory, Geriatric Research, Education and Clinical Center (GRECC), V. A. Wadsworth Hospital Center and the Department of Medicine, UCLA. Los Angeles, California 90073 Received September 20, 1978 An outbreak of parainfluenza type 1 (Sendai) virus infection in an aging mouse colony during our studies on immunologic aging permitted us to investigate the inverse temporal association between the decline in normal immune functions and the increased frequency of autoimmune manifestations. Emphasis was placed on the as yet unresolved issue of whether a decline in normal immune function precedes and, thus, predisposes to autoimmune disease (AID), or whether AID precedes and, thus, compromises normal immune function. Fifty-eight weight, cellular, activity, and autoimmune indexes were tested on each individual mouse before the viral infection and 63 indexes were tested up to 6 months afterward. The findings reported here show that a decline in the T cell, but not the B cell, component of the immune system precedes AID and that infection with a common respiratory virus can initiate AID in immunologically immature and immunodeficient aging individuals.

INTRODUCTION

Epidemiological studies have implicated common viruses in the initiation of certain human autoimmune diseases (AID) (1,2). However, because these studies were performed retrospectively, they lack preinfection immunological data on individuals who contracted viral infection and those who contracted viral infection followed by AID. For example, influenza viral infection has been reported to induce Coombs’-positive hemolytic anemias, basement membrane antibodies in the lungs and kidneys, and diabetes in humans (3-S). Moreover, the virus has been isolated from the kidney, heart, and other extrapulmonary tissues in some but not all fatal cases of human infections (6, 7). Although influenza infection is common and generally not particularly serious in healthy adults, it can be among elderly individuals as attested by actuarial findings of the influenza epidemics of 1957-58 and 1962-63 (6,7). A recent report of the World Health Organization (8) ranked influenza as the number one cause of morbidity in the Americas and Europe out of the 21 communicable diseases, and as the number 10th and 12th cause of mortality in the Americas and Europe, respectively. This is not surprising since normal immune functions can decline with age in both experimental animals and humans, and associated with the decline is a rise in the frequency of autoimmune manifestations (9- 11). The inverse temporal association of these two events suggests that they may be causally related. If so, autoimmunity may be intimately related to T-cell functional deficiency, since the decline in normal immune functions with age has been shown to be associated ’ This is publication No. 021 from GRECC. 301 0090-1229/79/030301-15$01.00/O Copyright All rights

0 1979 by Academic Press. Inc. of reproduction in any form reserved.

302

MARGUERITE

M.

B.

KAY

primarily with alteration in the T-cell population (12). One of the fundamental issues which have yet to be answered is whether the decline in normal immune function precedes and permits the development of AID, or whether AID precedes and compromises normal immune functions. These alternative possibilities are subjected to experimental scrutiny as a consequence of an unfortunate episode which occurred in the midst of our studies on immunologic aging. The episode was an epizootic infection which occurred in the aging mouse colony of the Gerontology Research Center of NIH. It was caused by parainfluenza type 1 virus (Sendai), which contains a hemagglutinin and neuraminidase as do influenza viruses (7). MATERIALS

AND METHODS

Animals. Adult (3-7 months), middle-aged (12-15 months), and old (19-24 months) CBA and CBA/T6T6 mice were used. They were bred in the Gerontology Research Center animal colony. Syngeneic CBA and CBA/T6T6 mice were selected for this study because they are considered to be resistant to AID. They do not develop Coombs’-positive red bIood cells (RBC) and antibodies to DNA and deoxyribonucleoprotein (DNP) even as late as 30 months of age, and neonatal thymectomy does not result in Coombs’ positivity even though about one-third of the thymectomized mice develop antibodies to DNA and DNP (13). Mice were thymectomized (T mice) at 4 weeks of age. Two weeks after thymectomy, they were exposed to 850R of X irradiation (300 KVP Biological Specimen X-ray irradiator, Ridge Instrument Co.) and then were given intravenously 5 x lo6 syngeneic bone marrow cells from 8-week-old donors (TXB mice). Electron microscopy. Scanning electron microscopy and transmission electron microscopy studies were performed 6-12 months postinfection. Washed RBC, thymus, lymph node, and spleen cells were fixed with 1% glutaraldehyde in Dulbecco’s phosphate-buffered saline (PBS) at 4”C, followed 2-3 hr later with 3% glutaraldehyde (14, 15), and processed for electron microscopy as described previously (16, 17). Positve controls consisted of RBC incubated with parainfluenza, type 1 virus (Sendai). Specimens were viewed with an Hitachi field emission scanning electron microscope with 3-nm resolution, or with an Hitachi 11 E transmission electron microscope. Immunojluorescent labeling of kidney sections. Kidneys were frozen immediately and 4-pm sections were cut, acetone fixed, washed three times in PBS, and overlaid with a 1:40 dilution of antimouse IgA, G, and M (Meloy Laboratories, Springfield, Va.). After a 30-min incubation at 37”C, sections were washed three times in PBS, and four drops of a 1: 1 solution of glycerol-PBS were added to the slides, which were covered with a coverslip and sealed with nail polish. Fluorescence was read and photographed with a Zeiss Universal microscope. Studies of the renal pathology including immunofluorescence, light histology, and transmission electron microscopy will be published elsewhere. Assays. A total of 63 indexes were determined on each mouse following infection (i.e., 7 weight, 31 cellular, 16 activity, and 9 autoimmune indexes). Experimentally determined weight indexes included body, lymph node, spleen, and thymus weight; calculated weight indexes derived from experimental determinations included percentage organ weight of body weight for lymph nodes, spleen, and thymus.

PARAINFLUENZA

AGING

AND

AUTOIMMUNE

DISEASE,

303

Cellular indexes included the following experimental determinations: cells per tissue, percentage dead cells at 4°C during routine processing for tissue culture, percentage T cells, percentage B cells, and percentage dead cells after a 30-min incubation at 37°C in culture media with 5% bovine serum albumin (BSA) for dispersed bone marrow, lymph node, spleen, and thymic cells. The following indexes were calculated from experimental determinations: cells per milligram wet weight for lymph nodes, spleen, and thymus; the number of B cells and T cells for bone marrow, lymph nodes, spleen, and thymus. Activity assays included the culture background (BKG) in counts per minute (cpm), a phytohagglutinin (PHA) dose-response (cpm) curve (0.03, 0.05, 0.10, 0.25,0.5, 1.0,2.0, and 3.0 pg) from which the dose eliciting the peak response was determined, the lipopolysaccharide (LPS) response (cpm) for lymph node and spleen cells, and the number of spleen colony-forming units (CFUS) per 5 x lo4 bone marrow cells (18, 19). The following indexes were calculated from experimental determinations: loglocpmPHA- loglOcpmBKG~ log,,cpmlps-log,,cpmB,,, loglo (cwhcpmBKd T hho(cpmws - cpm,,,) for dispersed lymph node and spleen cells, and the total CFU in the femoral bone marrow. Autoimmune indexes included: IgM and IgG Coombs’ titers and the percentage dead cells after a 30-min incubation at 37°C in culture medium containing 5% guinea pig complement for bone marrow, lymph node, spleen and thymus cells, anti-DNA and RNA antibody titers, and the hematocrit. Direct Coombs’ titers were assessed with serially diluted goat anti-mouse IgG or goat anti-mouse IgM (Meloy). For the determination of anti-DNA and anti-RNA autoantibodies, RNA and single-stranded DNA (Sigma Chemical Co., St. Louis, MO.) were conjugated to sheep red blood cells (SRBC) by adding 0.5 mg in 1.0 ml to the equivalent of 0.4 ml of packed SRBC in 9 ml of PBS. Glutaraldehyde (EM Sciences) was added over a period of 15 min so that the final concentration was 0.250/c, and the reaction allowed to continue for an additional 45 min, after which the SRBC were washed three times in PBS. Undiluted plasma from each mouse was incubated with either DNA or RNA-coated SRBC for 15 min at 37°C and 45 min at 4°C washed three times with PBS, and tested with a direct Coombs’ using anti-IgG and anti-IgM at doubling dilutions from 1:40 to 1: 10240. RESULTS Sendai Infection

The infection was brought into the Gerontology Research Center animal farm by a shipment of infected 5- to 6-week-old DBA/2 and C3H mice from Bethesda, Maryland. CBA mice had no detectable serum antibody titer to the virus prior to the infection, but subsequently had complement fixation titers of 1:40 to 1:80 (Microbiological Associates, Bethesda, Md.). All mice tested had either positive or “anticomplementary” complement fixation test results. Titers to other viruses did not change significantly. At the height of infection (2-3 weeks after the beginning of the outbreak), old mice appeared ill but the adult and middle-aged mice appeared normal. However, autopsies performed on randomly selected mice revealed pulmonary lesions typical of Sendai infection in mice and influenza infection in humans (20). Moreover, prospective recipient mice for the bone marrow CFUS that received 850 R died within 9 days, whereas none died from such radia-

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KAE

tion treatment prior to the injection. Three months after the infection, when postinfection studies were initiated, mice of all age groups appeared normal, and pulmonary lesions were not visible at autopsy. However, adult mice were still vulnerable to death following the radiation bone marrow treatment. Preinfectiorl

Age-Reluted

Indexes

The data were analyzed with a computer, employing the Pearson correlation coefficient for each index. Table 1 shows 14 indexes in CBA/T6T6 mice which correlated best with age. Ten of the 14 were thymic or thymic dependent and 9 of these 10 decreased with age. Four of 10 were B-cell indexes, of which two increased with age (splenic LPS response and the number of bone marrow B cells). Analysis of T-cell composition revealed that the number of T cells in the thymus decreased progressively with age, but the percentage did not decrease until after 12 months of age (Table 2). In contrast, the number of T cells in the lymph nodes remained stable until after 19 months when both number and frequency decreased. The number of T cells in the spleen did not change significantly with age. but the frequency showed a gradual decline which, however, was not significant. TABLE INDEXES

THAT

CORRELATE

BEST

WITH

I AGE

FOR

CBA/T6T6

MICE”

Pearson correlation coefftcient

P Valueb

Weight Thymus wt Percentage thymus wt of body wt

-0.88 -0.87

0.0001 0.0001

Cellular Number of T cells, thymus Percentage B cells, bone marrow Percentage B cells, lymph node Thymus cellularity Number of B cells, lymph node Cells per mg wet wt thymus Percentage dead cells. thymus Percentage T cells, thymus Number of T cells, lymph node

-0.92 0.86 -0.86 -0.85 -0.77 -0.67 0.54 -0.53 -0.50

0.0001 0.001 0.001 0.0001 0.01 0.0001 0.002 0.01 0.02

Activity’ Log,, (LPS - BKG), spleen Log,, (PHA - log,, BKG), lymph node Log,, (PHA - BKG), spleen

0.59 -0.57 -0.50

0.004 0.009 0.02

index

n The data for individual mice were correlated with chronological age. An index is included in the table if the absolute value of the Pearson correlation coefficient was between 0.50 and 1.OO.A correlation coefftcient of 1.0 indicates complete correlation between an index and age, and a correlation coefficient of - 1.O indicates a compiete inverse correlation. b The P value or significance probability of a correlation coefficient is the probability that a value of the correlation coefficient as large or larger in absolute value than the one calculated would have arisen by chance. were the two random variables (i.e., age versus the index) truly uncorrelated. c BKG. counts per minute of unstimulated background cultures; LPS, counts per minute of LPSstimulated cultures: PHA, counts per minute of PHA-stimulated cultures.

PARAINFLUENZA

CHANGE

AGING

AND

TABLE 2 COMPOSITION OF LYMPHOID

IN T-CELL Thymus

Age (months) 3 6 12

No. ( x R-8)

Lymph

Percentage’

53.8 (8.6)b 31.0 (5.7) 26.3

24

16.3 (3.1) 5.0 (3.1)

No. (x10-e)

nodes

(“2: 68 (7)

14.5 (1.4) 15.4 (3.5) 12.7

(3.6) 7.1 (2.9)

AGE Spleen

Percentage

(1.6) c”p,

305

DISEASE

TISSUES WITH

14.4

(3.2) 19

AUTOIMMUNE

:4: 52

(8) p61)

No. (X 10-y 39.8 (7 .O) 21.7 (2.7) 29.2 (8.5) 39.0 (10.2)

Percentage

:p, ::, 26

(2)

32.4 (7.0)

a Percentage of the total number of white cells which were T cells. Because the number of B cells did not decrease with age in most tissues, the results are not presented. b Numbers in parentheses are 2 SEM. Sample size, 6- 12 per group.

Figure la shows that thymic weight, the number of lymphoid cells per thymus, the number of cells per milligram of thymic tissue, and the percentage thymus weight of body weight decreased steadily with age. Thus, by 24 months these indexes were reduced to 39, 24, 48, and 35%, respectively, of those obtained at 6 months of age. The PHA response of lymph node and spleen cells decreased with age so that by 24 months of age they were 42 and 30%, respectively, of adult values (Fig. 2). Indexes Altered by Infection All indexes were affected by infection. The frequency and number of T cells could not be determined postinfection because the high frequency of cell death rendered a cytotoxicity assay invalid. Estimates of B-cell number and frequency were also unreliable. The PHA response decreased significantly following infection to levels approaching those of unstimulated cultures. CFUS could not be assessed because radiation resulted in death within 8 days in all adult mice tested. This occurred as late as 6 months postinfection in adult mice that appeared grossly normal. Cell viability. The frequency of cell death following routine processing at 4°C of lymphoid tissues for culture before infection was highest in cells dispersed from the thymus and bone marrow of old mice. The frequency of dead cells from the thymuses of 6- and 24-month-old mice were 1 and 22%, and from the bone marrow, 4 and 19% respectively (Table 3). Following infection, the frequency of dead cells after processing increased in all age groups, with the highest in old mice. Old cells were so labile that a significant increase in the frequency of dead cells was observed by merely incubating them in culture medium containing 5% BSA. This was not the case before infection (Fig. lb). Bone marrow cellularity (BMC). BMC, which remained relatively constant with age before infection (Fig. 3), increased with age following infection. Thus, in middle-aged and old mice, the numbers increased by 44 and 39%) respectively. An

306

MARGUERITE

M.

AGE

B.

IiA’I

(months)

FIG. 1. (a) Age-related changes in thymic indexes of CBAIT6T6 mice before parainfluenza type 1 virus infection. Thymus weight (0); number of Iymphoid cells per thymus (A); lymphoid cells per mg of thymic tissues (0, and percentage thymus weight of body weight (0) are plotted as the percentage of values obtained for 6month-old mice. Bars indicate standard error of the mean. (b) Frequency of cell death at 6 months postinfection at 37°C in Medium 199 with 5% bovine serum albumin. Cell viability was determined by the trypan blue dye exclusion technique. Bars indicate standard error of the mean. L, lymph node: T, thymus; S, spleen; and B, bone marrow.

6

12 AGE

18

24

(months)

FIG. 2. Relationship between PHA activity of lymph node and spleen cells before parainfluenza virus infection and Coombs’ titer 3 months after infection. Bars indicate standard error of the mean. Error bars are not shown for the Coombs’ titer because the standard error was zero.

PARAINFLUENZA

AGING

AND

TABLE THE EFFECT

Age (months) 6

OF SENDAI

INFECTION

L

T

I

1

20

(0)

24

3 OF LYMPHOID

CELLS

OF ACING

CBA/T6T6 MICE”

Postinfection

S 6 (1)

15

8

(2)

(2)

(1)

17 14

15 (3) 22

(2)

(6)

8 (1) 10 (4)

(2)

307

DISEASE

Preinfection

(2) 12

ON THE FRAGILITY

AUTOIMMUNE

8

B 4 (1)

7

(2) 0 19 (4)

L

T

S

B

36

23

(5)

(5)

14 (3)

(2)

27

16

(5)

(1)

10

31

31

12

15

6 (3) 6

co

(1)

(2)

(1)

35 (5)

24 (5)

37

cm

34 (4)

(1)

‘I Frequency of cell death (%) was determined after tissues were routinely processed for culture at 4°C. before they were washed because the percentage dead cells decreased following washing. Presumably, dead cells are lighter and, therefore, did not pellet during centrifugation and/or dead cells lysed during washing. L, lymph node; T, thymus; S, spleen: B, bone marrow. Numbers in parentheses are standard error of the mean.

increase of 44% was also detected among aging thymectomized and TXB mice. Further, mice that were 1 month old at the time of infection had an increase in BMC of 38% over preinfection values 5 months later. Autoimmunity. Prior to infection, CBA and CBA/T6T6 mice of all ages were Coombs’ negative, but following infection 100% of the 20- and 24-month-old mice were Coombs’ positive. The Coombs’ IgM titer also increased with age, i.e., 0 for 6 and lZmonth-old mice, 40 for 20-month-old mice and 80 for 24-month-old mice (Fig. 2). Mice that were 1 month old at the time of infection, and therefore im40 “z2 -

1

0 Ed

PRE- SENDAI POST - SENDAI

OLD FIG. 3. Bone marrow cellularity ( x lob) before parainfluenza infection. Bars indicate standard error of the mean.

virus infection and 3-6 months after

308

MARGUERITE

Sl.

H.

KA1

munologically immature, also had a Coombs’ IgM titer of 80 as late as 6 months after the outbreak. T and TXB mice also became Coombs’ positive. The results of these studies, summarized in Table 4, indicate that: adult and middle-aged mice that had not been rendered T-cell deficient did not develop Coombs’-positive hemolytic anemia; adult and middle-aged mice that were thymectomized, but whose peripheral T cells were not destroyed by radiation. became Coombs‘ positive but did not develop anemia; middle-aged TXB mice, which were deficient of all T cells save those immature precursors generated by the bone marrow. developed Coombs’-positive hemolytic anemia: and old mice which had not been immunologically manipulated developed Coombs’-positive hemolytic anemia. There was no increase in antibody titer to either DNA or RNA following the Sendai infection (results not presented). Immunofluorescent studies of the kidneys were performed 9- I? months postinfection. They showed both linear deposits of immunoglobulins along the glomerular basement membranes, and “lumpy” or granular deposits outside the basement membrane and in the mesangium of mice which were infected when they were one month old. Studies on old mice demonstrated granular deposits of immunoglobulins in the glomeruli. In contrast, none of the middle-aged mice examined had detectable immunoglobulin deposits in their glomeruli.

ION OF COO~IBS’-POSI.T~~F.

INITIA.I

VIRUS

Age (months)

IN IMMLIS~DEPRESSED AND IMMUNOLOGIC:ALLY

Time relative to Sendai infection

we

AUTOIMMUNE

TABLE 4 HEMOI.WI~

Asters

HY A PAKAINFI.L

ADLTI.I

.~ND MIDDLE-ACXJ DL:FICIEN~ OLD Mlcr”

Coombs’

ESJ..~ T\ pt

titer”

Treatment

Hematocrit

(% 1

post pre post

none none T’ T

0 0 0 33 -c 7

0 0 0 17 + 3

ND” 43.3 -t 0.3 ND 45.0 2 I.2

12 I? 14 IS

pre post post post

none none T TXB

0 0 60 IT 20 53 i 13

0 0 31-3 I? + 13

ND 46.0 f 0.6 43.0 + 0.6 ‘5.6 i- 3.7

19 20 18 24 24

pre post post pre post

none none TXB none none

0 40 5 0 120 2 40 0 80 i 0

0 0 13 _i 13 0 13 t 7

ND 30.0 2 3. I 30.7 t 3.9 ND 36.3 i 1.7

‘I Values are mean t standard error: sample size. 3 - I1 mice ’ Not determined. ’ T, thymectomized at 4 weeks of age: TXB. thymectomized reconstituted with bone marrow at 6 weeks of age.

I

MICE,

per group. at 4 weeks

of age and irradiated

and

PARAINFLUENZA

AGING

AND

AUTOIMMUNE

DISEASE

309

Scanning and Transmission Electron Microscopy Viral particles were not detected with either transmission electron microscopy or scanning electron microscopy on the surface of RBC from any of the experimental mice, even though they were easily visible on the surface of RBC which had been incubated with parainfluenza type 1 (Sendai) virus (Figs. 4a and b). However, viral-like particles were visible with transmission electron microscopy in RBC from the spleen and peripheral blood of TXB and old mice, but not in those of normal adult and middle-aged mice (Figs. 5a and b). DISCUSSION

Previous studies have shown that immunodeficiency and autoimmunity are often associated with each other in long-lived humans and mice, the frequency of individuals with serum autoantibodies increases with age, and the presence of autoantibodies in males is correlated with death from cardiovascular disease (9, 21, 22). Unfortunately, it has been difficult to determine whether a causal relationship exists between the two events, because these phenomenological studies were performed at a time when both immunodeficiency and autoimmunity were present. Other studies, in mice, have shown that certain T-cell activities decline early in the relatively short-lived NZB and A strain mice, which spontaneously develop AID. The decline precedes AID, and the onset of AID can be either accelerated by neonatal thymectomy or retarded by injection of syngeneic thymocytes (23-25). Although much knowledge has been gained by studies on these short-lived animal models, it is still uncertain whether they are appropriate models for the immunodeficiency states and AID occurring in aging long-lived animals and humans. The causes and mechanisms of the decline in normal immune functions and of the occurrence of autoimmune diseases may be different, particularly in view of the evidence that NZB mice carry, throughout life, Gross leukemia virus which participates in the development of AID (26, 27) and may contribute to the immunologic abnormalities. Evidence also indicates that the level of serum thymic activity declines 5-10 months earlier in NZB mice than in other mouse strains. Indeed, the thymus itself is abnormal, since it transfers AID when grafted into neonatally thymectomized mice of other, nonautoimmune, strains (28). Thus, it was felt that the issue of whether the decline in normal immune functions precedes and permits the development of AID, or whether AID precedes and compromises immune functions, should be addressed experimentally in long-lived strains of mice and epidemiologically in humans in a longitudinal manner. A unique opportunity to test this issue presented itself when our long-lived, aging CBA/T6T6 mice, whose age-related immunologic activities were being investigated, became infected with parainfluenza virus. A total of 63 immunologically related indexes was assessed in these aging mice 4- 12 months before they were naturally infected and 3 - 12 months after the infection. The results demonstrated that a decline in the T-cell component of the immune system precedes AID. This strongly supports the view that immunodeficiency precedes and predisposes an individual to AID. Another important conclusion is that infection by a common respiratory virus can initiate AID in immunologically old individuals and in immunologically immature young individuals, but not in immunologically mature adult individuals and immunologically adequate middle-aged individuals.

310

MARGUERITE

hl.

R.

IiA\

PARAINFLUENZA

AGING

AND

AUTOIMMUNE

DISEASE

311

312

MARGUERITE

M.

B.

K.41

It is interesting that mice infected within a month after birth, when they were immunologically immature, and adult and middle-aged thymectomized mice became Coombs’-positive following infection but failed to develop the Coombs’positive hemolytic anemia observed in middle-aged and old TXB mice and in normal old mice. Three possible explanations can be offered for these phenomena. The first, and perhaps least likely explanation, is that macrophages of TXB and old mice may be more efficient in their capacity to phagocytize opsonized RBC than those of adult and middle-aged thymectomized mice. The finding of Heidrick (29) that macrophages from old mice have more hydrolytic enzymes than macrophages from young mice, and findings of Perkins and Makinodan (30) that macrophages from old mice are more efficient in destroying sensitized RBC, support this possibility. The second possibility is that stem cells of middle-aged TXB and untreated old mice are less efficient than those of normal adult, and adult and middle-aged thymectomized mice, in their rate of generating new RBC. This possibility is suggested by findings that the ability of old stem cells to self-replicate and to expand clonally is significantly reduced compared to that of stem cells from adult mice (19, 3 1, 32). Evidence from kinetic studies, in which chemical insults such as phenylhydrazine, alkylating agents, and hydroxyurea were utilized to force stem cells to self-replicate, indicates that the more times stem cells self-replicate, the less they can differentiate (33-35). Likewise, kinetic experiments in which erythropoietin was utilized to induce stem cells to differentiate indicate that differentiation resulted in decreased proliferation or self-replication (36). This is consistent with the concept that stem cells either self-replicate or differentiate, depending upon environmental homeostatic signals (37). In contrast, studies in which the function of syngeneically transplanted old stem cells was assessed 3 - 12 months after transplantation indicate that old stem cells can reconstitute unstressed recipient mice to the same levels as those derived from young mice (38). However, these results should be taken with caution because they show that the variability of response of individuals ~r~ithinyoung and old stem cell groups was several fold greater than that between the two groups. Kinetics of stem cell proliferation and differentiation were not examined in this study. If one were to accept the results of this study together with those of the kinetic studies, they would indicate that either the generation time of old stem cells is increased and/or the differentiation time of their progeny, which are precursors of mature peripheral cells, is increased. Prolongation of stem cell selfreplication time, or of time in any of the differentiation compartments, could cause a decrease in the rate of production of mature RBC that could lead to anemia when old mice are stressed by an increased rate of destruction of RBC. Moreover, in the study reported here, both TXB and old mice developed Coombs’-positive autoimmune hemolytic anemia in spite of an increase in their BMC by 239% over values determined prior to infection when they were Coombs’ negative. That anemia is associated with increased BMC is consistent with the view that prolongation of both the stem cell self-replication time and the differentiation time can lead to a decreased rate of production of RBC. The third possible explanation is that along with a reduction in normal T-cell

PARAINFLUENZA

AGING

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AUTOIMMUNE

DISEASE

313

activities, the acitivity of a T cell subpopulation which is exerting a stimulatory effect on erythropoiesis is greatly reduced. This possibility is supported by the following evidence: hematopoiesis in heavily irradiated recipients reconstituted with parental stem cells is augmented by injection of T cells (39,40); and an anti-& sensitive regulatory cell which is present in normal adult bone marrow, spleen, and thymus is required for the promotion of differentiation of hematopoietic stem cells into erythrocytes (41). Since the regulatory cell may constitute ~1% of the total bone marrow (41), it is possible that enough of these cells have survived in T mice to allow them to maintain erythropoiesis at a level necessary to avoid anemia. Possibilities 2 and 3 need not be mutually exclusive. In fact, combining the two possibilities may offer the best explanation. Regardless of the mechanism responsible for the Coombs’-positive autoimmune hemolytic anemia in naturally aged and TXB aged mice, these experiments indicate that the production of RBC by elderly and immunodeficient individuals may be adequate provided they are not severely stressed by, for example, an infection. Such stress would reveal the deficit in their reserve of progenitor cells, as manifested by their inability to increase RBC production to compensate for increased destruction. Previous observations which have indicated decreased self-replication and differentiation capacity of stem cells utilized artificial insults such as radiation, transplantation, and chemicals to perturb homeostasis and force replication or differentiation (19, 3 l-35). In contrast, these experiments indicate, probably for the first time, that TXB and naturally aged individuals have limited progenitor cell reserve and cannot cope effectively with certain naturally occurring viral infections. They support the view that the kinetic limitations of aging stem cells discovered by these “artificial methods” do indeed exist in situ and become significant when the individual is stressed by disease. It follows then that limitations of stem cell reserve in elderly and immunodeficient individuals should be considered in the clinical setting when disease is present or when therapy with drugs which act on the hematopoietic system is contemplated. Since thymectomized, TXB and naturally old mice became Coombs’ positive, it would appear that T cells do not participate directly in AID following a natural parainfluenza virus infection. This study suggests that a deficit in the T-cell component of the immune system predisposes one to AID, possibly by permitting the persistence and circulation of parainfluenza type 1 (Sendai) virus. Persistence of this virus may lead to alteration of host cells and tissues in such a way as to promote a T-cell independent response by B cells. This interpretation is consistent with the report of Anderson et al., 1977 (42), that a cell mediated immune response participates in the eradication of Sendai virus, and with the data presented here and elsewhere indicating that B-cell activity remains relatively intact during aging [see (1 l)]. This explanation would account for autoimmunity in the presence of T-cell deficiency as observed both in animals and in humans (9, 42, 43). It also supports Allison’s hypothesis (44) that viruses can bypass the requirement for T cells responsive against self-antigens. According to this hypothesis, virus-specific antigens on the surface of host cells or host antigens in the envelopes of lipid-

314

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KA\’

containing viruses can induce the formation of antibodies that react with autologous cells. It should be emphasized however, that T-cell deficiency appears to be a necessary prerequisite for naturally occurring AID as described here. Finally, it should be mentioned that the appearance of Coombs’ positivity, antiglomerular basement membrane antibodies, and membranous glomerular nephritis occurred in mice that were infected within a month after birth when their normal T-cell immunologic functions are not fully developed, indicating that immunologically immature individuals can suffer severe sequelae following viral infection just as old individuals do. This consideration may be of importance to studies of the etiology of diseases such as juvenile diabetes. rheumatoid arthritis. and multiple sclerosis for which both autoimmune and infectious components are suspected [for review, see (211. In conclusion, these results suggest that T-cell immunodeficiency precedes and predisposes to AID, and that infection with a common respiratory virus can initiate AID in immunologically immature young and immunodefkient aging individuals. AID appears to be a potential but not obligatory sequelae of viral infections, and the host response, not the virus, appears to determine the course or pattern of the infection. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13.

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