- Email: [email protected]
In vitro transfer of specific reactivity to cytomegalovirus and candida to cord blood leukocytes with dialyzable leukocyte extracts
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
14,30&306
(1979)
In Vitro Transfer of Specific Reactivity to Cytomegalovirus and Candida to Cord Blood Leukocytes with Dialyzable Leukocyte Extracts’ M. C. SIRIANNI,“.’ *Department D&ruses
M. FIORILLI,“’ A. PANA.+ F. AIUTI$
M. PEZZELLA.$
AND
Medicine 111. tltlstitute oj’ Hygienr. flrlstifute of fnfrcfiorr.\ BDivision of Infectious Discuses II. Unir~ersiry of Rome. 00161 Rome, Ito!\
of lnternul
I. and
Received May 10, 1979 The ability of two preparations of dialyzable leukocyte extracts (DLE, formerly termed “dialyzable transfer factor”) to induce specific responsiveness by cord blood leukocytes (CBL) in the direct leukocyte migration inhibition (LMI) assay in vitro was studied. One donor of DLE (DLE-A) was immune to cytomegalovirus (CMV). as judged by positive LMI test, and negative to candida as judged by skin test. The other donor (DLE-B) was immune to both CMV and candida. Several concentrations of DLE and of antigen were tested. High concentrations of DLE nonspecifically inhibited leukocyte migration, while low concentrations had no effect on antigen-induced LMI. Antigenspecific conversion was produced by intermediate concentrations of DLE. Both preparations transferred reactivity to CMV. but only DLE-B transferred reactivity to candida ill Llit ro
INTRODUCTION
Since Lawrence’s first description of the effects of a leukocyte extract, which he termed “dialyzable transfer factor” (l), its ability to specifically transfer cellmediated immunity (CMI) to an uncommitted recipient has remained controversial, since the classical in Go transfer of specific skin reactivity from donor to recipient in man (2) was unsuccessful in the hands of some investigators (3, 4). New interest in the in ri~o transfer of skin reactivity arose after reports of the use of gnotobiotic animals as targets for human dialyzable leukocyte extracts (DLE) (5). Much more controversy has arisen from the use of in ~irro assays of DLE. The varied data presented at the Second International Workshop on Transfer Factor (6) pointed out the extreme difficulty of documenting a specific transfer of CM1 to uncommitted lymphocytes using the blastogenesis assay. However, at the Third International Workshop, the data presented seemed to confirm the sensitivity and reproducibility of in Gtro assays for the effects of DLE using the leukocyte migration inhibition (LMI) test (e.g., 7, 8). It is generally agreed that DLE contains at least one component with a nonspecific adjuvant activity, which magnifies the responses to antigens (9) and mitogens (10) in vitro and acts as an immunostimulant in lvi~o (i.e., increases E ’ This work has been supported by a grant from the National munodeficiencies. 4 To whom requests for reprints should be addressed. 300 0090-1229/79/l 103Ol-07$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
Council
of Research for Im-
SPECIFIC
TF ACTIVITY
IN
LMI
301
rosettes, mixed leukocyte reaction, and mitogenic responses) (11, 12), as well as a component which induces and/or enhances antigen-specific CM1 (13). In the present article we report that DLE is effective in conferring to cord blood leukocytes reactivity against cytomegalovirus (CMV) and candida antigens, using the direct LMI assay. Evidence is presented that the specificity of the transfer for candida is dependent on the reactivity of the DLE donor. MATERIALS
AND
METHODS
Indicator cells. DLE was tested on the leukocytes of eight healthy, full-term newborns. Heparinized (preservative-free heparin, 10 U/ml) cord blood was obtained under sterile conditions; erythrocytes were allowed to sediment and the leukocyte-rich plasma was harvested. The leukocytes were washed three times and resuspended at a concentration of 6 x lo6 cells/ml in RPM1 1640 containing penicillin (10 IU/ml), streptomycin (10 pg/rnl), and 5% fetal calf serum (complete RPM1 1640). All cultures were set up within 2 hr of delivery. DLE preparations. Leukocytes were collected from donors with an IBM continuous flow centrifuge. After performing total and differential counts, equal volumes of sterile water were added to the cell suspensions. The mixtures were then frozen-thawed 10 times and ultrafiltered through Amicon Diallow PM-30 membranes. The ultrafiltrates were adjusted in normal saline to a concentration of 10’ lymphocytes/ml (calculated on the basis of the original cell numbers), then passed through a Millipore filter (0.22 pm), placed in ampules, and lyophilized. For testing, each ampule was brought to the original volume with sterile pyrogen-free distilled water. DLE donors. DLE was prepared from two healthy adults, both with cellular immunity to CMV; for the latter, both donors had a positive direct LMI test (31); donor A had migration indexes (see below) ranging from 40 to 70% at various antigen concentrations; donor B ranged from 40 to 60%. The donors differed in regard to their skin test responses to candida: Donor A had no response while donor B showed a 22-mm induration area 48 hr after the intradermal injection of 0.1 ml of a 1: 100 dilution of Dermatophyton (Hollister Stier) in saline. Both had normal E-rosette levels and did not excrete CMV in their urine. Incubation with DLE. Each cord blood cell suspension was incubated with several concentrations of one of the two DLE preparations. One ampule of DLE was redissolved and diluted to 1: 10, 1: 100, and 1: 1000 with sterile saline; 0.1 ml of each dilution was added to 1 ml of leukocyte suspension (6 x lo6 cells/ml), to obtain a final concentration of DLE of, respectively, 106, lo”, and lo4 lymphocytes/ml. Then 0.1 ml of sterile saline was added to the control cultures. Control and DLE cultures were incubated for 1 hr at 37°C in 5% CO, and the cells were then used without further washing in the direct LMI test. In preliminary experiments we preincubated the cells for up to 3 hr and found no difference in the results, except for a decreasing viability of PMN and thus we used the shortest reported (incubation) time. Leukocyte migration inhibition. The LMI test was performed according to Federlin ef al. (14). In brief, siliconized capillary tubes (20 ~1, Drummond Microcaps) were filled with the cell suspensions. The capillary tubes were then heatsealed and centrifuged at 200g for 10 min. The tubes were cut at the cell-fluid
302
SIRIANNI
ET- AL.
interface and the capillary stumps were placed on the floor of plastic disposable migration chamber plates (Sterilin Ltd.) and held in place by a drop of silicon grease. The chambers were filled with complete RPM1 1640 alone (control) or containing the following antigens: CMV at the concentration listed below: candida (Dermatophyton, Hollister Stier) diluted to 1: 10 and 1: 100. All the assays were set up in quadruplicate. After 20 hr of incubation at 37°C in 5% CO, the migration areas were blindly evaluated by planimetry. For each combination (DLE alone, antigen alone, DLE + antigen) the migration indexes (MI) were calculated as follows: M, = Average area of migration in presence of DLE t antigen x loo. Average are of migration in control medium The activity of DLE was evaluated by comparing (by paired I test) the MI for DLE alone versus the MI for DLE + antigen. E rosettes (sheep r-osettes). Lymphocytes were isolated from the leukocyte-rich plasma on a Ficoll-Isopaque gradient (15), washed twice, and resuspended at 4 x 10” cells/ml in complete RPM1 1640. To 1 ml was added 0.1 ml of the usual DLE dilutions or of normal saline. The mixtures were incubated for 1 hr at 37°C in 5% CO,. The E rosettes were then enumerated according to the method of Jondal cf (11. (16). Virologiccrl
studies. Living CMV (strain AD169), serially propagated on human embryo diploid cells, was used in the LMI assay. The virus was diluted in complete RPM1 1640 at concentrations of 3000, 300. 30, and 3 mean tissue culture doses/ml (TCD,,,/ml). Congenital CMV infection was excluded in all neonates by the absence of viruria and specific tests for serum IgM antibodies to CMV (indirect immunofluorescence with a minimum serum dilution of 1116). RESULTS
The results of the LMI test against CMV in the absence or presence of the two DLE preparations are shown in Fig. 1. In all cases, when the cells were cultured without DLE, no migration inhibition was observed in the presence of CMV. When the cells were preincubated with DLE at the highest concentration (10” lymphocyte equivalent/ml), both DLE-A and DLE-B had a marked inhibitory effect, which was not increased by the addition of antigen. At intermediate concentrations (10”/ml), DLE-B alone showed no activity, while DLE-A slightly inhibited the cell migration; however, a strong inhibition was observed with both preparations after the addition of CMV (DLE-A P < 0.0025: DLE-B P -c 0.005). Variations of the antigen dose did not significantly affect the results. At the lowest concentration of DLE (104/ml) no migration inhibition was observed either in the presence or absence of CMV. A similar dose-effect relationship with the concentration of DLE was observed when using candida as antigen (see Table 1). At the concentration of 10Yml DLE-A (candida negative) had no effect, while DLE-B (candida positive) induced a significant (P < 0.005) migration inhibition in the presence of the antigen. Finally, the dialysates did not affect the percentage of E rosettes at any concentration. Representative values for E rosettes were 57 + 9.8% in control cultures
SPECIFIC TF ACTIVITY
DLE-A I I
X
0” z
IN LMI
I
I
-1 .-. i*+*
I
T
-? /-! i
303
.-
-.-
i
1
i I
DLE-B
I 3000
DLE alone
I
300
DLE +CMV
I
I
30
3
(TCD so/ml)
No DLE ----DLE 104/ml DLE 10S/ml .-.-. DLE 105/ml FIG. 1. Effect of DLE on the response of neonatal leukocytes to CMV antigen in the direct LMI test. Means ? SD are reported. *, Not significant; **, P < 0.0025: ***, P < 0.005. l
l
and 58.6 + 5% after incubation DLE-B).
l
l
l
l
with DLE at 1OVml (pooled data from DLE-A
and
DISCUSSION
Several previous reports have failed to demonstrate specific in vitro transfer of CM1 to uncommitted lymphocytes by DLE (6). Ahern and Sanderson were unable
304
SlRlANNl
ET
TABLE EFFEC,F
OF DLE
ON THY
CANDIDA
RESPONSE
ANUGEN
AL.
I OF Nt0~41.41
IN .THE DIRH
L~UKWYIFS
I LMI
‘10
TESI”
DLL: Control 1:lO”
1:lOO
10”iml
I o”/ ml
0
DLE-A
91 k6.3
1: 10 1:lOO 0 I: 10 -___-- ___-. ~~~~. ~. 90.7 60.8 6Y.4 86.3’ 78.1 76.5 +7.1 -1-25.3 tlY.I t13.7 i-21.2 +-14.X
DLE-B
106.7 -+7.3
105. I 53 24.8 t11.7
79.2’ 70.8 224.6 226.1
Y6.4 40.6” i9 I-13.Y
lO’/ml I:100
0
79.x X0.X L 14.1 rtl7.6 41.7 -? 14.7
99. I +3.3
I: 10
I : 100
75.5 ~23.3
70.9 i24.2
106.3 -7Y,1 I____.-.
103.8 -75.1 I_.
0 Results expressed as migration indexes (mean t SD). * Dilution of candida added. Zero dilution indicates that no candida was added. ” Not significant. d P < 0.005.
to show any effect of DLE of known specificity on antigen-induced lymphocyte transformation under optimal culture conditions ( 17). In another study, Miiller et a’/. unsuccessfully used cord blood cells as targets for DLE in a blastogenesis assay (18). These experiments strongly suggested that antigen-triggered lymphocyte DNA synthesis could not be induced de nova by DLE. In contrast, Basten and Croft (19) reported in ~~itro conversion of negative adult lymphocytes to positive in an antigen-specific, donor-dependent fashion, using macrophage migration inhibition as the criterion: however they were unable to reproduce this experiment with cord blood cells as targets (20). This implied that the effect in adults was an amplification of a minimal preexisting reactivity; alternatively, it can be hypothesized that adult and neonatal lymphocytes differ in their sensitivity to DLE, and that the concentration of DLE used was insufficient to activate newborn lymphocytes. In general, antigen-specific conversion seems to be more clearly demonstrable in vitro by migration inhibition assays than by lymphocyte proliferation (20-23), even though, in the hands of some investigators, the two methods have given similar results (24). This observation has several possible explanations: i.e., production of lymphokines and DNA synthesis could be properties of different cell populations (25), only one of which is a target for DLE, or the biological event of lymphokine production could be more easily (or uniquely) influenced by DLE. The fact that this is a real phenomenon is also supported by the dissociation of MIF production and lymphocyte transformation in response to antigens in patients with chronic mucocutaneous candidiasis (26, 27) and Wiskott-Aldrich syndrome (28) treated with DLE. Our observation of a direct inhibitory effect of DLE on leukocyte migration in vitro is in agreement with other reports (13). Cord blood lymphocytes in general can be considered as uncommitted, although some degree of reactivity has been demonstrated for several antigens, including herpes simplex virus (29). In our studies the lack of a baseline LMI response to CMV and candida, together with the absence of serum IgM antibody to CMV and
SPECIFIC TF ACTIVITY
IN LMI
305
viruria, can reasonably be considered as indicating lack of immunological commitment to these antigens. However, whether true “virginity” of neonatal cells to such widespread antigens does indeed exist remains a serious concern. Our finding that DLE did not increase the percentage of E rosettes in normal peripheral blood lymphocytes without prior enzyme treatment is in agreement with data reported by others (30). We previously demonstrated that direct LMI correlates well with serological results for CMV antibodies (31) and with the skin test for candida (unpublished data); thus, it seems to us a suitable model for in Gtro testing of the biological activity of DLE. A definite answer to this point wil! ,,ome from experiments employing other less common antigens, and from studies of the activity of purified fractions of DLE with this test system. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Lawrence, H. S.. 3. Cl/n. Invest. 34, 219, 1955 Lawrence, H. S., Harvey Leer. 68, 239, 1974. Spitler, L. E., Levin, A. S., and Fudenberg, H. H., C/in. Zrnmunobiol. 2, 153, 1974. Neidhart, J. A., Christakis, N., Metz, E. N., Balcerzak, S. P.. and LoBuglio, A. F., J. A//erg> C/in. Zmmunol. 61, 115, 1978. Eichberg, J. W.. Steele, R. W., Kalter, S. S., Kniker, W. T., Heberling, R. L., Eller, J. J., and Rodriguez, A. R.. Cell Zrnmunol. 26, 114, 1976. Lawrence, H. S., In “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 741-753, Academic Press, London, 1976. Borkowsky, W., and Lawrence, H. S., J. C/in. Hemarol. Oncol. 8, 112. 1978 (abstract). Wilson, G. B., and Fudenberg, H. H., J. C/in. Hematol. Oncol. 8, 112, 1978 (abstract). Hamblin, A. S.. Dumonde, D. C., and Maini, R. M., C/in. Exp. Zmmunol. 23, 290, 1976. Hamblin, A. S., Dumonde, D. C., and Maini, R. M., C/in. E.Y~. Zmmunol. 23, 303, 1976. Valdimarsson, H., Hambleton, G., Henry, K., and McConnell, I., C/in. Exp. Zmmunol. 10, 141, 1974. Wybran, J., Levin, A. S., Spitler, L. E., and Fudenberg, H. H., N. Engl. /. Med. 288,710, 1973. Wilson, G. B., et al.. Trans. Assoc. Amer. Phys. 91, 294, 1978. Federlin, K., Maini, R. N., Russell, A. S., and Dumonde, D. C., J. C/in. Parho/. 14, 533, 1971. Boyurn, A., Stand. J. C/in. Lab. Znraest. 21 (Suppl. 97), 1, 1968. Jondal, M., Holm, G., and Wigzell, H., .Z. Exp. Med. 136, 207, 1972. Ahem. T., and Sanderson, C. J., C/in. Exp. Zmmunol. 23, 499, 1976. Mtlller, M. R., Grob., P. J., and Hitzig, W. H., Znt. Arch. Allergy Appl. Zmmunol. 54, 269, 1977. Basten, A., and Croft, S., In “Immunological Engineering” (D.W. Jirsch, Ed.), pp. 83-120, MTP Press, Lancaster, 1978. Basten, A., Croft, S., and Edwards, J., Zn “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 75-84, Academic Press, London, 1976. Baram, P., and Condulis, W., Transplanr. Proc. 6, 209, 1974. Salaman, M. R., Immunology 26, 1069. 1974. Dunnick, W. A., and Bach, F. H., Proc. Nat. Acad. Sci. USA 72, 4573, 1974. Dunnick, W. A., and Bach, F. H., J. Zmmunol. 118, 1944, 1977. Rocklin, R. E., J. Zmmunol. 110, 674, 1973. Kirkpatrick, C. H., and Smith, T. K., Ann. Znt. Med. 80, 310, 1974. Arala Chavez, M. P., Horsmanheimo, M., Goust., J. M., and Fudenberg, H. H., In “Immunological Engineering” (D. W. Jirsch, Ed.), pp. 35-82, MTP Press, Lancaster, 1978. Spitler, L. E., Levin, A. S., Stites, D. F., Fudenberg, H. H., Pirofsky, B., August, C. S., Stiehm, E. R., Hitzig, W. H., and Gatti, R. A., J. C/in. Invest. 51, 3216, 1972. Russell, A. S., C/in. Exp. Zmmunol. 22, 457, 1975.
306
SIRIANNI
ET
AI..
30. Kahn, A., Garrison, O., Thometz, D., Hill, J. M., I/I “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher. A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 335-340. Academic Press, London, 1976. 31. Fiorilli, M., Sirianni, M. C., Spaziani, A., Aiuti. F.. and Pani. A.. La,lc,er 1, 780. 1978.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
14,30&306
(1979)
In Vitro Transfer of Specific Reactivity to Cytomegalovirus and Candida to Cord Blood Leukocytes with Dialyzable Leukocyte Extracts’ M. C. SIRIANNI,“.’ *Department D&ruses
M. FIORILLI,“’ A. PANA.+ F. AIUTI$
M. PEZZELLA.$
AND
Medicine 111. tltlstitute oj’ Hygienr. flrlstifute of fnfrcfiorr.\ BDivision of Infectious Discuses II. Unir~ersiry of Rome. 00161 Rome, Ito!\
of lnternul
I. and
Received May 10, 1979 The ability of two preparations of dialyzable leukocyte extracts (DLE, formerly termed “dialyzable transfer factor”) to induce specific responsiveness by cord blood leukocytes (CBL) in the direct leukocyte migration inhibition (LMI) assay in vitro was studied. One donor of DLE (DLE-A) was immune to cytomegalovirus (CMV). as judged by positive LMI test, and negative to candida as judged by skin test. The other donor (DLE-B) was immune to both CMV and candida. Several concentrations of DLE and of antigen were tested. High concentrations of DLE nonspecifically inhibited leukocyte migration, while low concentrations had no effect on antigen-induced LMI. Antigenspecific conversion was produced by intermediate concentrations of DLE. Both preparations transferred reactivity to CMV. but only DLE-B transferred reactivity to candida ill Llit ro
INTRODUCTION
Since Lawrence’s first description of the effects of a leukocyte extract, which he termed “dialyzable transfer factor” (l), its ability to specifically transfer cellmediated immunity (CMI) to an uncommitted recipient has remained controversial, since the classical in Go transfer of specific skin reactivity from donor to recipient in man (2) was unsuccessful in the hands of some investigators (3, 4). New interest in the in ri~o transfer of skin reactivity arose after reports of the use of gnotobiotic animals as targets for human dialyzable leukocyte extracts (DLE) (5). Much more controversy has arisen from the use of in ~irro assays of DLE. The varied data presented at the Second International Workshop on Transfer Factor (6) pointed out the extreme difficulty of documenting a specific transfer of CM1 to uncommitted lymphocytes using the blastogenesis assay. However, at the Third International Workshop, the data presented seemed to confirm the sensitivity and reproducibility of in Gtro assays for the effects of DLE using the leukocyte migration inhibition (LMI) test (e.g., 7, 8). It is generally agreed that DLE contains at least one component with a nonspecific adjuvant activity, which magnifies the responses to antigens (9) and mitogens (10) in vitro and acts as an immunostimulant in lvi~o (i.e., increases E ’ This work has been supported by a grant from the National munodeficiencies. 4 To whom requests for reprints should be addressed. 300 0090-1229/79/l 103Ol-07$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
Council
of Research for Im-
SPECIFIC
TF ACTIVITY
IN
LMI
301
rosettes, mixed leukocyte reaction, and mitogenic responses) (11, 12), as well as a component which induces and/or enhances antigen-specific CM1 (13). In the present article we report that DLE is effective in conferring to cord blood leukocytes reactivity against cytomegalovirus (CMV) and candida antigens, using the direct LMI assay. Evidence is presented that the specificity of the transfer for candida is dependent on the reactivity of the DLE donor. MATERIALS
AND
METHODS
Indicator cells. DLE was tested on the leukocytes of eight healthy, full-term newborns. Heparinized (preservative-free heparin, 10 U/ml) cord blood was obtained under sterile conditions; erythrocytes were allowed to sediment and the leukocyte-rich plasma was harvested. The leukocytes were washed three times and resuspended at a concentration of 6 x lo6 cells/ml in RPM1 1640 containing penicillin (10 IU/ml), streptomycin (10 pg/rnl), and 5% fetal calf serum (complete RPM1 1640). All cultures were set up within 2 hr of delivery. DLE preparations. Leukocytes were collected from donors with an IBM continuous flow centrifuge. After performing total and differential counts, equal volumes of sterile water were added to the cell suspensions. The mixtures were then frozen-thawed 10 times and ultrafiltered through Amicon Diallow PM-30 membranes. The ultrafiltrates were adjusted in normal saline to a concentration of 10’ lymphocytes/ml (calculated on the basis of the original cell numbers), then passed through a Millipore filter (0.22 pm), placed in ampules, and lyophilized. For testing, each ampule was brought to the original volume with sterile pyrogen-free distilled water. DLE donors. DLE was prepared from two healthy adults, both with cellular immunity to CMV; for the latter, both donors had a positive direct LMI test (31); donor A had migration indexes (see below) ranging from 40 to 70% at various antigen concentrations; donor B ranged from 40 to 60%. The donors differed in regard to their skin test responses to candida: Donor A had no response while donor B showed a 22-mm induration area 48 hr after the intradermal injection of 0.1 ml of a 1: 100 dilution of Dermatophyton (Hollister Stier) in saline. Both had normal E-rosette levels and did not excrete CMV in their urine. Incubation with DLE. Each cord blood cell suspension was incubated with several concentrations of one of the two DLE preparations. One ampule of DLE was redissolved and diluted to 1: 10, 1: 100, and 1: 1000 with sterile saline; 0.1 ml of each dilution was added to 1 ml of leukocyte suspension (6 x lo6 cells/ml), to obtain a final concentration of DLE of, respectively, 106, lo”, and lo4 lymphocytes/ml. Then 0.1 ml of sterile saline was added to the control cultures. Control and DLE cultures were incubated for 1 hr at 37°C in 5% CO, and the cells were then used without further washing in the direct LMI test. In preliminary experiments we preincubated the cells for up to 3 hr and found no difference in the results, except for a decreasing viability of PMN and thus we used the shortest reported (incubation) time. Leukocyte migration inhibition. The LMI test was performed according to Federlin ef al. (14). In brief, siliconized capillary tubes (20 ~1, Drummond Microcaps) were filled with the cell suspensions. The capillary tubes were then heatsealed and centrifuged at 200g for 10 min. The tubes were cut at the cell-fluid
302
SIRIANNI
ET- AL.
interface and the capillary stumps were placed on the floor of plastic disposable migration chamber plates (Sterilin Ltd.) and held in place by a drop of silicon grease. The chambers were filled with complete RPM1 1640 alone (control) or containing the following antigens: CMV at the concentration listed below: candida (Dermatophyton, Hollister Stier) diluted to 1: 10 and 1: 100. All the assays were set up in quadruplicate. After 20 hr of incubation at 37°C in 5% CO, the migration areas were blindly evaluated by planimetry. For each combination (DLE alone, antigen alone, DLE + antigen) the migration indexes (MI) were calculated as follows: M, = Average area of migration in presence of DLE t antigen x loo. Average are of migration in control medium The activity of DLE was evaluated by comparing (by paired I test) the MI for DLE alone versus the MI for DLE + antigen. E rosettes (sheep r-osettes). Lymphocytes were isolated from the leukocyte-rich plasma on a Ficoll-Isopaque gradient (15), washed twice, and resuspended at 4 x 10” cells/ml in complete RPM1 1640. To 1 ml was added 0.1 ml of the usual DLE dilutions or of normal saline. The mixtures were incubated for 1 hr at 37°C in 5% CO,. The E rosettes were then enumerated according to the method of Jondal cf (11. (16). Virologiccrl
studies. Living CMV (strain AD169), serially propagated on human embryo diploid cells, was used in the LMI assay. The virus was diluted in complete RPM1 1640 at concentrations of 3000, 300. 30, and 3 mean tissue culture doses/ml (TCD,,,/ml). Congenital CMV infection was excluded in all neonates by the absence of viruria and specific tests for serum IgM antibodies to CMV (indirect immunofluorescence with a minimum serum dilution of 1116). RESULTS
The results of the LMI test against CMV in the absence or presence of the two DLE preparations are shown in Fig. 1. In all cases, when the cells were cultured without DLE, no migration inhibition was observed in the presence of CMV. When the cells were preincubated with DLE at the highest concentration (10” lymphocyte equivalent/ml), both DLE-A and DLE-B had a marked inhibitory effect, which was not increased by the addition of antigen. At intermediate concentrations (10”/ml), DLE-B alone showed no activity, while DLE-A slightly inhibited the cell migration; however, a strong inhibition was observed with both preparations after the addition of CMV (DLE-A P < 0.0025: DLE-B P -c 0.005). Variations of the antigen dose did not significantly affect the results. At the lowest concentration of DLE (104/ml) no migration inhibition was observed either in the presence or absence of CMV. A similar dose-effect relationship with the concentration of DLE was observed when using candida as antigen (see Table 1). At the concentration of 10Yml DLE-A (candida negative) had no effect, while DLE-B (candida positive) induced a significant (P < 0.005) migration inhibition in the presence of the antigen. Finally, the dialysates did not affect the percentage of E rosettes at any concentration. Representative values for E rosettes were 57 + 9.8% in control cultures
SPECIFIC TF ACTIVITY
DLE-A I I
X
0” z
IN LMI
I
I
-1 .-. i*+*
I
T
-? /-! i
303
.-
-.-
i
1
i I
DLE-B
I 3000
DLE alone
I
300
DLE +CMV
I
I
30
3
(TCD so/ml)
No DLE ----DLE 104/ml DLE 10S/ml .-.-. DLE 105/ml FIG. 1. Effect of DLE on the response of neonatal leukocytes to CMV antigen in the direct LMI test. Means ? SD are reported. *, Not significant; **, P < 0.0025: ***, P < 0.005. l
l
and 58.6 + 5% after incubation DLE-B).
l
l
l
l
with DLE at 1OVml (pooled data from DLE-A
and
DISCUSSION
Several previous reports have failed to demonstrate specific in vitro transfer of CM1 to uncommitted lymphocytes by DLE (6). Ahern and Sanderson were unable
304
SlRlANNl
ET
TABLE EFFEC,F
OF DLE
ON THY
CANDIDA
RESPONSE
ANUGEN
AL.
I OF Nt0~41.41
IN .THE DIRH
L~UKWYIFS
I LMI
‘10
TESI”
DLL: Control 1:lO”
1:lOO
10”iml
I o”/ ml
0
DLE-A
91 k6.3
1: 10 1:lOO 0 I: 10 -___-- ___-. ~~~~. ~. 90.7 60.8 6Y.4 86.3’ 78.1 76.5 +7.1 -1-25.3 tlY.I t13.7 i-21.2 +-14.X
DLE-B
106.7 -+7.3
105. I 53 24.8 t11.7
79.2’ 70.8 224.6 226.1
Y6.4 40.6” i9 I-13.Y
lO’/ml I:100
0
79.x X0.X L 14.1 rtl7.6 41.7 -? 14.7
99. I +3.3
I: 10
I : 100
75.5 ~23.3
70.9 i24.2
106.3 -7Y,1 I____.-.
103.8 -75.1 I_.
0 Results expressed as migration indexes (mean t SD). * Dilution of candida added. Zero dilution indicates that no candida was added. ” Not significant. d P < 0.005.
to show any effect of DLE of known specificity on antigen-induced lymphocyte transformation under optimal culture conditions ( 17). In another study, Miiller et a’/. unsuccessfully used cord blood cells as targets for DLE in a blastogenesis assay (18). These experiments strongly suggested that antigen-triggered lymphocyte DNA synthesis could not be induced de nova by DLE. In contrast, Basten and Croft (19) reported in ~~itro conversion of negative adult lymphocytes to positive in an antigen-specific, donor-dependent fashion, using macrophage migration inhibition as the criterion: however they were unable to reproduce this experiment with cord blood cells as targets (20). This implied that the effect in adults was an amplification of a minimal preexisting reactivity; alternatively, it can be hypothesized that adult and neonatal lymphocytes differ in their sensitivity to DLE, and that the concentration of DLE used was insufficient to activate newborn lymphocytes. In general, antigen-specific conversion seems to be more clearly demonstrable in vitro by migration inhibition assays than by lymphocyte proliferation (20-23), even though, in the hands of some investigators, the two methods have given similar results (24). This observation has several possible explanations: i.e., production of lymphokines and DNA synthesis could be properties of different cell populations (25), only one of which is a target for DLE, or the biological event of lymphokine production could be more easily (or uniquely) influenced by DLE. The fact that this is a real phenomenon is also supported by the dissociation of MIF production and lymphocyte transformation in response to antigens in patients with chronic mucocutaneous candidiasis (26, 27) and Wiskott-Aldrich syndrome (28) treated with DLE. Our observation of a direct inhibitory effect of DLE on leukocyte migration in vitro is in agreement with other reports (13). Cord blood lymphocytes in general can be considered as uncommitted, although some degree of reactivity has been demonstrated for several antigens, including herpes simplex virus (29). In our studies the lack of a baseline LMI response to CMV and candida, together with the absence of serum IgM antibody to CMV and
SPECIFIC TF ACTIVITY
IN LMI
305
viruria, can reasonably be considered as indicating lack of immunological commitment to these antigens. However, whether true “virginity” of neonatal cells to such widespread antigens does indeed exist remains a serious concern. Our finding that DLE did not increase the percentage of E rosettes in normal peripheral blood lymphocytes without prior enzyme treatment is in agreement with data reported by others (30). We previously demonstrated that direct LMI correlates well with serological results for CMV antibodies (31) and with the skin test for candida (unpublished data); thus, it seems to us a suitable model for in Gtro testing of the biological activity of DLE. A definite answer to this point wil! ,,ome from experiments employing other less common antigens, and from studies of the activity of purified fractions of DLE with this test system. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Lawrence, H. S.. 3. Cl/n. Invest. 34, 219, 1955 Lawrence, H. S., Harvey Leer. 68, 239, 1974. Spitler, L. E., Levin, A. S., and Fudenberg, H. H., C/in. Zrnmunobiol. 2, 153, 1974. Neidhart, J. A., Christakis, N., Metz, E. N., Balcerzak, S. P.. and LoBuglio, A. F., J. A//erg> C/in. Zmmunol. 61, 115, 1978. Eichberg, J. W.. Steele, R. W., Kalter, S. S., Kniker, W. T., Heberling, R. L., Eller, J. J., and Rodriguez, A. R.. Cell Zrnmunol. 26, 114, 1976. Lawrence, H. S., In “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 741-753, Academic Press, London, 1976. Borkowsky, W., and Lawrence, H. S., J. C/in. Hemarol. Oncol. 8, 112. 1978 (abstract). Wilson, G. B., and Fudenberg, H. H., J. C/in. Hematol. Oncol. 8, 112, 1978 (abstract). Hamblin, A. S.. Dumonde, D. C., and Maini, R. M., C/in. Exp. Zmmunol. 23, 290, 1976. Hamblin, A. S., Dumonde, D. C., and Maini, R. M., C/in. E.Y~. Zmmunol. 23, 303, 1976. Valdimarsson, H., Hambleton, G., Henry, K., and McConnell, I., C/in. Exp. Zmmunol. 10, 141, 1974. Wybran, J., Levin, A. S., Spitler, L. E., and Fudenberg, H. H., N. Engl. /. Med. 288,710, 1973. Wilson, G. B., et al.. Trans. Assoc. Amer. Phys. 91, 294, 1978. Federlin, K., Maini, R. N., Russell, A. S., and Dumonde, D. C., J. C/in. Parho/. 14, 533, 1971. Boyurn, A., Stand. J. C/in. Lab. Znraest. 21 (Suppl. 97), 1, 1968. Jondal, M., Holm, G., and Wigzell, H., .Z. Exp. Med. 136, 207, 1972. Ahem. T., and Sanderson, C. J., C/in. Exp. Zmmunol. 23, 499, 1976. Mtlller, M. R., Grob., P. J., and Hitzig, W. H., Znt. Arch. Allergy Appl. Zmmunol. 54, 269, 1977. Basten, A., and Croft, S., In “Immunological Engineering” (D.W. Jirsch, Ed.), pp. 83-120, MTP Press, Lancaster, 1978. Basten, A., Croft, S., and Edwards, J., Zn “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher, A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 75-84, Academic Press, London, 1976. Baram, P., and Condulis, W., Transplanr. Proc. 6, 209, 1974. Salaman, M. R., Immunology 26, 1069. 1974. Dunnick, W. A., and Bach, F. H., Proc. Nat. Acad. Sci. USA 72, 4573, 1974. Dunnick, W. A., and Bach, F. H., J. Zmmunol. 118, 1944, 1977. Rocklin, R. E., J. Zmmunol. 110, 674, 1973. Kirkpatrick, C. H., and Smith, T. K., Ann. Znt. Med. 80, 310, 1974. Arala Chavez, M. P., Horsmanheimo, M., Goust., J. M., and Fudenberg, H. H., In “Immunological Engineering” (D. W. Jirsch, Ed.), pp. 35-82, MTP Press, Lancaster, 1978. Spitler, L. E., Levin, A. S., Stites, D. F., Fudenberg, H. H., Pirofsky, B., August, C. S., Stiehm, E. R., Hitzig, W. H., and Gatti, R. A., J. C/in. Invest. 51, 3216, 1972. Russell, A. S., C/in. Exp. Zmmunol. 22, 457, 1975.
306
SIRIANNI
ET
AI..
30. Kahn, A., Garrison, O., Thometz, D., Hill, J. M., I/I “Transfer Factor. Basic Properties and Clinical Applications” (M. S. Ascher. A. A. Gottlieb, and C. H. Kirkpatrick, Eds.), pp. 335-340. Academic Press, London, 1976. 31. Fiorilli, M., Sirianni, M. C., Spaziani, A., Aiuti. F.. and Pani. A.. La,lc,er 1, 780. 1978.