Use of “Synovial” Cell Cultures in the Search for Virus in Rheumatoid Arthritis

By DENYS K. FORD AND JANG 0. OH

Synovial cells obtained from rheuma- toid and nonrheumatoid subjects were grown in culture for periods up to 5 months. No spontaneous degeneration, suggestive of latent viral infection, was observed in the rheumatoid cultures. No significant difference could be dem- onstrated between multiplication of Newcastle disease virus in rheumatoid and nonrheumatoid cultures. No virus- interfering or -inactivating activity could be demonstrated in the culture fluid from rheumatoid synovial cultures.

Cellulas synovial ab subjectos rheuma- toide e non-rheumatoide esseva man- tenite in culturation durante periodos de usque a 5 menses. Nulle degenera- tion spontanee de character suggestion- ante tun latente infection viral esseva observate in le culturas rheumatoide. Nulle significative differentia esseva demonstrabile inter culturas rheuma- toide e non-rheumatoide con respecto a1 multiplication de virus de morbo de Newcastle. Nulle activitate de inter- ferentia o de inactivation de crescentia de virus poteva esser demonstrate in le liquido cultural ab culturas de rheu- matoide cellulas synovial.

EW SYSTEMATIC ATTEMPTS to isolate a virus from the synovial fluid F of patients with rheumatoid arthritis have been reported. In this labora- tory no cytopathic changes have been observed in primary African green monkey kidney, primary human amnion and subcultured human embryonic lung cultures inoculated with synovial fluid from 20 patients with rheumatoid arthritis and observed for up to 5 blind passes at 3-week intervals. In order to search for indirect evidence that a virus might be involved in rheumatoid arthritis we have studied synovial cells cultured in vitro. These cultures have been observed for spontaneous degeneration which might suggest the presence of a latent virus. In addition, experiments have been performed to attempt the demonstration of a virus by use of the interference phenomenon which was applied recently with success to the initial recognition of the common cold virus,l the rubella virus2 and a virus in a cell line derived from Burkitt’s lymphoma.3 In these situations cultured cells proved resistant to infection by an experimentally inoculated challenge virus because the cells already con- tained a virus which “interfered with the infection of the cell by the challenge virus.

SYXOVIAL CELL CULTURE METHODS A. Nonrheumatoid synovial cells were obtained from the knee joints of cadavers within

4 hours of death by the method of Fraser and Catt.4 After warming the knee joints with a plastic bag containing hot water, the bynovial cavity was washed out with 2 changes of Earle’s basic salt solution and then injected with 25-30 ml. volumes of 0.25 per cent

DENYS K. FORD, M.D., Associate Professor, Director of Canadian Arthritis and Rheuma- tism Society Research Unii, Department of Medicine, University of British Columbia, Vancouver, R . C., C a d . JANG 0. OH, M.D., PH.D., Assistant Professor, Department of Pathology, University of British Columbia, Vancouver, B . C., Canada.

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ARTHRITIS AND RHEUMATISM, VOL. 8, No. 6 (DECEMBER), 1965

1048 FORD AND OH

trypsin (Difco 1:250) in basic salt solution at a pH of 7.6. Two to three changes of trypsin were left in the joint cavity for 30 minutes each and collected in 50 ml. centrifuge tubes. Aftcr centrifuging at 1,000 R.P.M. for 10 minutes and discarding the supernatant, the deposit was resuspended in culture medium (Eagle’s Basal Medium with 20 per cent fctal bovine serum and penicillin). A cell count was then made and the suspension diluted with the same medium to give a count of 500,000 cells per ml. before distribu- tion of 1.5 ml. to screw cap tubes.

B. Rheumatoid synovial cells were obtained from the knee joints of patients with classical rheumatoid arthritis by the following method. One hour after the patient had received 50 mg. of diphenhydramine, a # l 6 needle with a &way stop cock was intro- duced into the knee and the joint cavity washed with several changes of normal saline from a 250 ml. intravenous bottle connected by tubing to the stop cock. Twenty to 50 ml. of Armour’s Tryptar diluted in basic salt solution to 2,000 units per ml. were in- jected into the joint and left in situ for 45 minutes before being reaspirated. Three fur- ther similar washings with Tryptar were made and all the washings collected. Finally the knee joint was irrigated twice with the original saline and 50 mg. of hydrocortisone acetate suspension injected. The Tryptar and final saline washings were centrifuged and the cell deposit resuspended in medium; the resulting suspension was counted, diluted to a cell concentration of 2 million cells per ml. before distribution of 1.5 ml. to screw cap culture tubes. The procedure was well tolerated by the patients and resulted in only a slight increase of swelling and pain in the joint for 12 to 24 hours.

C. Synovial cells from both nonrheumatoid and rheumatoid subjects were obtained from synovial tissue removed at arthroplasty, arthrodesis, or open arthrotomy, the non- rheumatoid subjects having osteoarthritis or chondro-malacia of the patella; operations for simple meniscectomy of the knee did not provide enough synovial tissue. The re- moved tissue was minced and exposed to 0.25 per cent trypsin while agitated by a magnetic stirrer. The cell suspension from 2 to 5 trypsinizations of 30 to 40 minutes each was Centrifuged, resuspended in medium, counted, diluted to 0.5 and 2 million cells per ml. respectively and 1.5 ml. distributed to screw cap culture tubes. Cultures obtained from rheumatoid subjects always contained a great number of acute inflammatory cells and thus were initiated with greater cell counts than the nonrheumatoid cultures. The medium was changed on the second day of culture and at 2 to 3 day intervals there- after. The inflammatory cells soon disappeared due to their failure to adhere to the glass, with subsequent removal at medium changes. The pH of the medium was ad- justed between 7.2 to 7.5 as necessary. Twenty per cent fetal bovine serum was always used to initiate cultures but well established cultures in which rapid growth was not required were switched to 10 per cent serum. Cell strains were subcultured through the use of 0.25 per cent trypsin.

SYNOVIAL CELL CULTURE RESULTS

Table 1 shows the number of cell strains obtained by the three technics and with each method actively multiplying healthy-looking cell cultures usually developed in 1 to 3 weeks and provided 6 to 20 tubes per strain. The cultures continued to multiply for over 2 months and some remained viable for 8 months, but in most cases, the cultures were used for experiments within a few weeks after their initiation. No continuous cell lines were de- veloped and it appeared that, after 5 to 8 months, the cultures usually propagated less and slowly degenerated. At maximal growth it was possible to subculture the cells every 10 to 14 days and double the number of tubes available. Only a few of the nonrheumatoid cultures derived from cadavers were used for the viral experiments described here, the others being used for

“SYNOVIAL” CELL CULTURES 1049

Table 1.-Results of Cell Culture Procedures No. Used for NDV* No. of Strains No. Viable

Source of Cultures Initiated Cultures Experiments

Nonrheumatoid

Nonrheumatoid

Rheumatoid intact

Rheumatoid operative

morgue cases 83 69 10

operative tissue 14 14 14

joint “Tryptar m e t h o d 10 6 4

tissue 12 12 12

Total: 119 101 40

“Newcastle disease virus.

preliminary studies and also for cytological and immunological experiments. Those chosen for viral studies were typical cultures and selected only because they were in culture and available as controls when the experiments on rheumatoid cultures were performed.

The cells cultured by each method were fibroblastic in appearance, the majority being spindle shaped although in some cultures polygonal cells predominated. The synovial origin of the cells was often suggested by an increase in viscosity of the supernatant medium; however, attempts to dis- tinguish these cell cultures from other fibroblast and monocyte cultures by the application of Alcian blue and alkaline and acid phosphatase staining pro- cedures were not successful. Thus no proof was available that these were indeed “synovial cell” cultures. The viability of the cells in the cultures was tested by both neutral red and trypan blue staining and it was found that healthy cells overwhelmingly predominated.

The cultures from rheumatoid patients were closely observed for up to 5 months for spontaneous degeneration but none was seen, the cultures proliferating and appearing as healthy as the nonrheumatoid cultures. Similarly, no morphological differences were observed between rheumatoid and non- rheumatoid cultures with the light microscope.

Experiments to Detect Resistance to Virus of Synovial Cells Derived from Rheumatoid and Nonrheumatoid Subjects Primary synovial cell cultures obtained from both the trypsinization of

intact knee joints by the methods previously described and also from the trypsinization of surgically removed tissue were used for these experiments. Newcastle disease virus ( N D V ) was chosen as a challenging virus and its multiplication in the two groups of cells was compared. Following inoculation of lo4 plaque forming units (PFU) of NDV into duplicate tube cultures, the cells were incubated for 1 hour at 37 C. for virus adsorption. They were then washed and exposed for 1 hour at 37 C. to 50 hemagglutination-inhibiting units of rabbit anti-NDV serum to eliminate virus unadsorbed to the cells. After the cells had been again washed with phosphate buffered saline (PBS)

1050

I

5 -

4 - - E

D! VI a

? r 3 -

SYNOVIAL CULTURES FROM INTACT JOINTS

5 -

4 -

FORD AND OH

SYNOVIAL CULTURES FROM OPERATIVE TISSUE

- RHEUMATOID CULTURES .......... NON RHEUMATOID CULTURES

i ... . . i .. . . . . .. i

..,.

.................. I'.. ..... ,.d .... I ....... .......... 8 . . . . . . . . . . . . . . . :. %. .... ,.'; .............

_...-

3

2

1 , " A I N O N " *

0 4 6 8 0 2 4 6 8 TIME IN DAYS TIME IN DAYS

Figs. 1 and B.--Growth curves of Newcastle disease virus in synovial cell cultures from intact joints (fig. 1) and operative tissue (fig. 2 ) .

they were incubated at 37 C. in fresh culture medium. Virus infectivity in specimens collected at successive 2-day intervals was titrated by the plaque count method of Dulbeccoj on monolayer cultures of chicken embryo fibro- blasts.

Ten nonrheumatoid and 4 rheumatoid cultures were available from those derived from the trypsinization of intact synovial cavities. Figure 1 shows that there was no significant difference in the multiplication of NDV between the 2 cell groups, the majority supporting virus multiplication to a titer of lo4 PFU/ml. or greater; the points on the graph represent the average of 2 duplicate tubes. However, one rheumatoid and 2 non-rheumatoid cultures did not support virus multiplication to a titer of 102 PFU/ml.

Fourteen nonrheumatoid and 12 rheumatoid cultures were available from those derived from the trypsinization of operative tissue. Figure 2 shows the growth curves obtained. Whereas the duplicate tubes of the previous experi- ment had given good agreement in virus titer, we found that there was considerable variation between the virus titers of the duplicate tubes of some cultures obtained from operative tissue. The graphs therefore represent the highest titer obtained on the days indicated.

It is evident from figure 2 that there was a tendency for NDV to multiply to a greater extent in the nonrheumatoid cultures and this is confirmed by table 2 in which the cultures have been labelled susceptible or resistant de- pending on whether or not the virus titer of lo2 PFU/ml. was achieved. The diff erenees were not statistically significant however.

1051 “SYNOVIAL” CELL CULTURES

Table 2.-Comparison of Ability of Rheumatoid and Nonrheumatoid Cultures to Support the Multiplication of NDV

Total Susceptible* ltesistantt

Intact R.A. 4 3 1 joints Non-R.A. 10 8 2

tissues Non-R.A. 14 10 4 Operative R.A. 12 4 8

““Susceptible” indicates that virus was produced to a titer of 102 PFU/ml. or greater. +“Resistant” indicates that virus was produced to a titer below 102 PFU/ml.

Experiments to Detect a Virus-interfering Agent or a Virus-inactivating Agent in the Primary Coltwe Fluid of Rheumatoid Synovial Cells Because the previous experiments had indicated that rheumatoid cultures

might be somewhat more resistant to NDV than nonrheumatoid synovial cells, the following two types of experiments were performed.

Tube cultures of HeLa, WISH human anmion or nonrheumatoid synovial cells were inoculated with 0.2 ml. of culture fluid from primary synovial cell cultures. Thirteen rheumatoid and 8 nonrheumatoid culture fluids were tested. One day later the cells were washed once with PBS and challenged with 10 TCID;,o (50 per cent tissue culture infectious dose) of vaccinia, vesicular stomatitis or Newcastle disease viruses. The cells were then observed to detect any resistance in the cells pre-exposed to the rheumatoid cell culture fluid. A complete destruction of the cells by the viruses was, however, observed in all tubes, indicating the absence of any interfering property.

To detect any NDV-inactivating substance in culture fluid from primary synovial cells, known amounts of NDV were added to 2 day culture fluid of both nonrheumatoid and rheumatoid synovial cells. Residual virus was then titrated at various time intervals and compared to the virus titer in fresh culture medium inoculated with the same amount of ~ i r u s . The rates of inactivation were the same in all 3 groups.

DISCIJSSION AXD SUMMARY

No difference could be demonstrated in the multiplication of Newcastle disease virus between rheumatoid and nonrheumatoid synovial cultures when these cultures were initiated from intact synovial cavities. When the cultures were initiated from operatively removed synovial tissue NDV appeared to multiply less well in rheumatoid than in nonrheumatoid cultures. The differ- ences were not striking, however, and the number of cases studied was small; moreover, there was considerable variation in virus titer between duplicate tubes in this experiment. The variabilities of this complicated series of technics are too great to permit the definition of minor differences. No spontaneous degenerations were seen in rheumatoid synovial cells to suggest the presence of a latent virus and this conforms to the recent experience of BartfeId.6 No virus-interfering or inactivating activity could be demonstrated in the culture fluid from rheumatoid synovial cultures.

FORD AND OH 1052

The negative findings in these experiments do not, however, exclude the possibility that viruses might be involved in the pathogenesis of rheumatoid arthritis.

ACKNOWLEDGMENTS The authors are grateful for the excellent technical assistance of Miss H. Ireland, Mrs.

The work was supported by Medical Research Council Grant MA-1432 and Federal J. MacDonald and Miss E. Swait.

Health Grant 609-7-40.

REFERENCES 1. Hitchcock, G., and Tyrrell, D. A. J.:

Some virus isolations from common colds. 11. Virus interference in tissne cultures. Lancet 1:237, 1960.

2. Parkman, P. D., Buescher, E. L., and Artenstein, M. S.: Recovery of ru- bella virus from army recruits. Proc. SOC. Exp. Biol. Med. 111:225, 1962.

3. Henle, G., and Henle, W.: Evidence for a persistent viral infection in a cell line derived from Burkitt’s lymphoma. J. Bact. 89:252, 1965.

4. Fraser, J. R. E., and Catt, K. J.: Hu- man synovial cell culture: use of a new method in a study of rheumatoid arthritis. Lancet 2: 1437, 1961.

5. Dulbecco, R.: Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proc. Nat. Acad. Sc. 38:747, 1952.

6. Bartfeld, H.: Rheumatoid arthritic and non-rheumatoid synovium in cell cul- ture. Ann. Rheumat. Dis. 24:31, 1965.