Cytotoxic and soluble mediator responses by complement (C3) receptor-bearing lymphocytes from patients with B-cell immunodeficiency diseases

CLINICAL

IMMUNOLOGY

ANDIMMUNOPATHOLOGY

6,279-288(1976)

Cytotoxic and Soluble Mediator Responses by Complement (C3) Receptor-Bearing Lymphocytes from Patients with B-Cell lmmunodeficiency Diseases BRUCE F. MACKLER'~~ ELLEN

PEGGY

RICHIE,~

‘Laboratory of Immunology, Dental Health Science Center at Houston; Vmmunology, Department of Pediatrics. Pediatrics, M. D. Anderson Hospital

O’NEILL~, NALINI MUKHOPADHYAY AND JOHN MONTCOMERY~

,2

Science Institute, The University of Texas 2Sections of HematologyDncology and Baylor College of Medicine; 3Department of and Tumor Institute. Houston, Texas 77025

Received August 7, 1975 Peripheral blood lymphoid subpopulations from congenital (X-linked) agammaglobulmemic and selective IgA-dehcient patients were assessed for nonspecific cytotoxicity and lymphotoxin (LT) effector responses. Complement receptor-bearing lymphocyte-mediated cytotoxicity and culture supernatant LT responses were quantitated using Wr-labeled human melanoma target cells. All patients appeared to have normal numbers of T and B cells as defined by E and EAC resetting while agammaglobulinemic patients were deficient in cells bearing surface membrane-associated immunoglobulin (SmIg). T cells and enriched B cells obtained by T-cell depletion gave negligible nonspecific cytolytic responses. In contrast, complement (C3) receptorbearing lymphocytes (CRL) from B-cell-immunodeficient patients and normal donors gave significant cytolytic and supematant lymphotoxin responses when activated by EAC rosetting. Evidence is presented that CRL from immunodeficient patients with total or partial B-cell deficiencies can still express C3 receptor-induced nonspecific cytotoxicity and lymphotoxin effector responses. The absence of serum and membrane-associated immunoglobulins did not impair the expression of C3 receptor-induced responses.

INTRODUCTION Evidence is accumulating that bone marrow-derived (B) lymphocytes can manifest cell-mediated effector responses previously ascribed solely to thymusderived (T) cells. Non-T-cell-mediated direct cytotoxicity toward certain tumors has been demonstrated in the absence of antibody (1,Z). Human B cells have been shown to undergo blastogenesis to mitogens and antigens as well as produce soluble mediators (3-7). The binding of membrane complement receptors (C3) was found to induce soluble mediator nonspecifically (3). Subsequent studies indicated that cross-linking of other membrane receptors also evoked cellular effector responses (8). Human monocyte chemotactic factors (CTX), induced by C3 receptor binding, were shown by isoelectrofocusing to be physicochemically identical to CTX derived from mitogen-stimulated T cells (9). In addition to generating CTX, C3 receptor binding induces nonspecific cytotoxic responses independent of antibody and involving lymphotoxin (LT) activities (10, 11). In view of these

5 Reprint requests to: Dr. Bruce F. Mackler, Dental Science Institute, The University of Texas Health Science Center, P. 0. Box 20068, Houston, Tex. 77025. Copyright 0 1976 by Academic Press. Inc. All tights of reproduction in any form reserved.

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ET AL.

reports, patients with B-cell immunodeficiency syndromes were studied to determine whether the expression of C3 receptor-induced responses requires the presence of surface immunoglobulin-bearing (SmIg) cells. Separated lymphoid subpopulations obtained from the peripheral blood of congenital (X-linked) agammaglobulinemic and selective &A-deficient patients were assessed for nonspecific effector responses. C3 receptor-bearing lymphocytes (CRL) from patients were activated by EAC rosetting. The activated CRL manifested cytolytic and soluble mediator responses in the absence of SmIg-bearing cells. The cytolytic reactivities observed with CRL from B-cell-immunodeficient patients were comparable to those levels obtained with activated CRL from normal donors. METHODS AND MATERIALS Patient population. The diagnoses of immunodeficiency diseases for all patients were based on the clinical information shown in Table 1. Congenital (X-linked) agammaglobulinemia was diagnosed by family histories, recurrent disease patterns, clinical and immunological evidence, and the occurrence of the disease prior to age 3 years. The serum and salivary immunoglobulin levels shown were determined at diagnosis or, in the case of B. H., immediately prior to y-globulin therapy. Blood for cell-mediated effector responses was obtained prior to plasma therapy (W.W.) or at least 2-3 months after the last y-globulin injection (SM. and B.H.). The diagnosis of agammaglobulinemia with rheumatoid arthritis-type syndrome was made for B. H. on the clinical findings as well as a history of severe recurrent infections and rheumatoid arthritis symptoms. Two patients with selective IgA deficiency were also examined. All immunodehciency patients had normal levels of circulating lymphocytes and phagocytic monocytes; the latter were determined by neutral red dye uptake. MNC were incubated for 30 min with neutral red dye in isotonic saline and the percentage of labeled cells was quantitated. Blood was also obtained from normal donors for comparative studies. Lymphoid subpopulation fractionation and characterization. Mononuclear cells (MNC) were separated from 20-40 ml of heparinized whole blood by Ficoll-Hypaque gradient centrifugation, giving a 65-85% recovery of MNC. Lymphoid subpopulations were separated by sequential rosetting as previously described (6, 10). T cells forming sheep erythrocyte (E) rosettes (E rosetteforming cells, E-RFC) were hrst separated from MNC and the remaining non-ERFC were incubated with E coated with IgM (19s) anti-E globulin and mouse complement (EAC) to form EAC rosette-forming cells (EAC-RFC). CRL subpopulations were also separated using zymosan-complement (ZC) reagent formed via the alternate complement pathway in the absence of antibody. ZC were prepared by heating zymosan (lOa particles/ml; Sigma Chemical Company, St. Louis, MO.) for 1 hr at 100°C and incubating them with an equal volume of fresh mouse serum. The washed ZC complexes were reacted with non-E-RFC and the ZC-RFC separated by Ficoll-Hypaque gradient centrifugation. The distribution of surface membrane immunoglobulin (SmIg) was assessed with a fluorescein-conjugated polyvalent rabbit anti-human immunoglobulin serum (C520, Meloy Labs., Springfield, Va.). Details of the method used to prepare culture supernatants have been fully described elsewhere (3, 6, 10).

CRL

RESPONSES

OF

IMMUNODEFICIENT

PATIENTS

0

0

0

0

i% d

0

+

+

+

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ET AL.

Cytotoxicity and lymphotoxin (LT) assays. Lymphocyte cytotoxicity and lymphotoxin (LT) activities were quantitated by the 51Cr assay previously described (10, 11). Human malignant melanoma @H-l) target cell monolayers cultured in Eagle’s minimal essential medium (MEM) plus 10% heat-inactivated fetal calf serum were labeled with lOO$Zi of sodium chromate (51Cr, ICN Pharmaceuticals, Irvine, Calif.; sp act, 300 G/g) for 18 hr at 37°C in 5% CO, in air. Labeled cells were washed three times, trypsinized, and adjusted to lo4 cells/ml. The appropriate number (104- 106) of lymphocytes or volume of culture supernatant was added to tubes containing 1 ml of cells making a total volume of 2 ml. The mixtures were incubated for 24 hr, and l-ml supernatant samples were assessed for radioactivity. The total 51Cr incorporation by lo4 target cells was determined by water lysis and spontaneous 51Cr release by control culture supernatants. The percentage of 5iCr release alas determined using the formula:

experimental 51Cr release - spontaneous 51Cr release total 51Cr incorporated - spontaneous 51Cr release

x 100.

The total 51Cr-incorporated levels by lo4 target cells averaged 6584.6 ? 803.9 cpm (standard error) by water lysis while spontaneous 51Cr-release levels averaged 290.7 + 31.5 cpm (SE). RESULTS Characterization

of Lymphocytes

The presence of cell surface markers on MNC from immunodeficient patients is compared with control subjects in Table 2. The MNC counts for whole blood are given along with the percentage recoveries of MNC after Ficoll-Hypaque graTABLE DISTRIBUTION

OF MEMBRANE

Total MNC in blood Patients (N)” (MNC/mm3) Congenital agammaglobulinemia S.M. 2385 (2) (1650-3120)6 W.W. (1) 3021 B.H. 2939 (2) (2X0-3317) IgA deficient C.M. 2253 (2) (1680-2823) Normals (8) 2941 t 661d

MARKERS

2

ON LYMPHOCYTES

(43?7) 69 (727) 64 (53-75) 73.1 t 9.3

IMMIJNODEFICIENT

-

Mononuclear (% MNC)b

Recovery of MNC by Ficoll-Hypaque

cm

FROM

E-RFC

EAC-RFC

75.6 (75-76) 53 59 (55-63) 60 (40-79)

~~

PATIENTS

~~~ ~-~

cells EAC-RFC (%)b

+SmIg

(N)

+Smlg

11 6-16) 29

2.8 (1.5-t) 8 0.9 (O-l .8)

(1)

3.2

(1) (1)

24.0 5.6

12 (10-12)

9.5 (4-15)

-

n.d.’

61.4 -t 9.7 14.8 t- 5.6 16.3 _t 3.9 (4) 90.3 k 4.0

a (N), number of experiments. * Percentages calculated by counting at least 200 cells per assay. ‘SmIg-surface membrane immunoglobulin staining cells. * Range of values; where N was >3, the mean k standard deviation is shown. p Not done.

CRL

RESPONSES

OF

IMMUNODEFICIENT

283

PATIENTS

dient centrifugation. This loss of MNC (14-35%) during Ficoll-Hypaque separation made the use of absolute numbers tenuous; therefore, the data are given as percentages of rosette-forming and immunoglobulin-bearing cells in the recovered MNC population. Despite congenital B-cell abnormalities, the percentages of TABLE 3 THE CYTOTOXICITY OF LYMPHOID SUBPOPULATIONS FROM NORMAL DONORS AND IMMUNODEFICIENT PATIENTS Cytotoxicity”

Donors and lymphoid cells

Target celhlymphocyte ratio

Normals E-RFC Non-E-RFC Non-E-RFC + Zd Non-E-RFC + Autologous Serum (1: 10) EAC-RFC (CRL) ZC-RFC (CRL) Dissociated CRL

Wr Release (av % 2 SE)

1:lO 1:20 1:20 1:20

(9) (4) (3) (4)

1.2 +- 0.3

1:20 1:20 I:20

(8) (3) (3)

17.5 2 2.1 23.5 2 3.1 22.1 ” 4.0

P valuesr compared to normal non-E-RFC (0.7 -c 0.4)

-2 n.s.’ n.s.

0.7 t 0.4 5.5 k 3.2

1.5 ? 0.6


compared

normal EAC-RFC (17.5 2 2.1) Congenital (X-linked) S.M. E-RFC EAC-RFC W.W. E-RFC EAC-RFC B.H. E-RFC EAC-RFC IgA deficient C.M. E-RFC EAC-RFC L.S. E-RFC EAC-RFC

agammaglobulinemia 1:lOO I:20 1:lO

(2)

1:lOO

-

(3) (3)

1.0 2 0.2 11.2 ? 2.8 11.2 -r- 3.6

n.s! n.s.

1:20

(1) (1)

0.2 9.6

ILS.

1:lOO 1:20

(1) (1)

1.2 8.7

n.s.

1:lOO 1:20 I:10

(2)

1:lOO 1:20 1:lO

(3) (4) (4)

a Melanoma target cell @H-l). b (N), number of experiments. c P values obtained by Student’s t test. d Z, zymosan. p Not significant.

(3) (3)

1.5 t- 0.5 14.3 ? 1.6 13.5 2 2.0

1.5 * 0.3 10.9 * 1.1 8.8 k 1.4

-

n.s. n.s.

n.s. n.s.

to

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ET AL

E-RFC and EAC-RFC from MNC of these immunodeficiency patients were comparable to those of normal donors. These patients had no evidence of lymphopenia or abnormal shifts in cell types as assessedby morphological examination. However, the percentages of SmIg + cells in MNC t?om the agammaglobulinemic patients were lower than those of normal donors. Of particular interest was the distribution of SmIg-staining cells in the EAC-RFC subpopulation. Purified EAC-RFC from normal donors contained 86-94% SmIg + cells whereas EACRFC from agammaglobulinemic patients had reduced levels (3.1-24%) of SmIg + cells. Cytotoxicity

of Lymphoid

Subpopulations

Lymphoid subpopulations from normal donors assayed for nonspecific cytotoxicity are shown in Table 3 using melanoma target cells (SH-1); comparable results have been obtained with human lung fibroblast (10). The E-RFC and a population enriched for B cells (non-E-RFC) prepared by T-cell depletion gave negligible nonspecific cytotoxic responses even when tested at target cell:lymphocyte ratios of 1:200, l:lOO, and 150. Non-E-RFC incubated with Z also gave no cytotoxicity. In contrast, CRL rosetted with either zymosan-complement (ZC) or EAC reagents gave significantly higher (P
LYMPHOCYTES

*oo,CQO

300,CQO 200,GOO loO,ooO 10.000

TARGET

CELL

: LYMPHOCYTE

I.000

500

RATIOS

FIG. 1. CRL-mediated cytotoxicity at various target ceklymphocyte ratios. The target cells were held constant (10,000 cells/culture) while CRL from normal donors varied from 4OO;Qooto 5Qo cells/ culture. CRL used were obtained from normal donors. Each point represents the sextuplet replicates; the vertical lines show standard error.

CRL

RESPONSES

OF IMMUNODEFICIENT

285

PATIENTS

induced appreciable cytoxicity (12.8%). CRL were dissociated from adherent EAC with anti-C3 globulin (3, 6) and tested for cytotoxicity. These experiments with dissociated CRL excluded the possibility that either erythrocytes, erythrocyte membrane fragments, or the EAC complex were bridging between the effector CRL and the target cell. The removal of adherent EAC did not diminish cytotoxic activities of the dissociated CRL. Nonspecific cytotoxicity of lymphoid subpopulations from immunodeficient patients is shown in Table 3. EAC-RFC obtained from congenital agammaglobulinemic patients gave appreciable cytotoxicity (8.7 - 11.4% Vr release) at 1:20 ratios as well as 1:lO. The limited number of CRL available from 20-40 ml of blood precluded doing dose-response curves. These cytotoxicity levels were not significantly (ns.) different from those obtained with EAC-RFC from normal donors. EAC-RFC from the two patients with selective IgA deficiencies gave cytotoxic responses of 10.9 - 14.3%51Crrelease which were not signifkantly different from normals. In contrast, E-RFC from these two groups of immunodeficient patients gave negligible responses. These data suggested that CRL from patients

LYMPHOTOXIN

(LT)

DERIVED

FROM

TABLE 4 E-RFC AND EAC-RFC

LYMPHOID

Lymphotoxin Donors and lymphoid subpopulation Normal donors E-RFC E-RFC + PHA (1 mg/ml) EAC-RFC

Sup test dilution 1:3 1:6 1:3 1:6 1:3 1:6

W)b (7) (10) (3)

05) (6) (1-a

Avg. % YIr Release t S.E. 1.3 1.2 2.6 4.4 8.6 7.5

2 0.2 t 0.1 k 0.2 -c 0.9 2 0.6 k 0.3

SUBPOPULATION

activity” P valueSe compared to E-RFC (1.3 2 0.2)

n.s.d
Congenital (X-linked) S.M. E-RFC EAC-RFC W.W. E-RFC EAC-RFC IgA deficiency CM. E-RFC EAC-RFC

agammaglobulinemia 1:3 1:6

(2) (2)

1.3 t 0.2 6.3 f 0.7

n.s.

1:3 1:3

(1) (1)

0.3 10.9

n.s.

1:3 1:6

(1)

(2)

2.3 5.2 t 0.7

n.s.

a Melanoma target cell @H-l). * (N), number of experiments. r P values were obtained by Student’s r test. d Not significant.

286

MACKLER

ET AI

with total or partial B-cell immunodeficiencies cytotoxic responses.

could still manifest

C3-induced

Lymphotoxin (LT) Derived from E-RFC and EAC-RFC Subpopulations Since EAC-rosetted normal human B cells are known to produce soluble mediators (3, 6, 9) which include LT (lo), we investigated whether EAC-RFC from immunodeficient patients also produced LT. For comparison, supernatants from normal lymphoid subpopulations were assessed in the 51Cr release cytotoxicity assay for LT activities (Table 4). E-RFC from normal donors stimulated with phytohemagglutinin (PHA) produced only 2-3.5 times more LT than unstimulated control cultures. In contrast, supernatants from EAC-RFC cultures contained significant LT levels, seven to eight times higher than that of control E-RFC cultures. Supernatants of EAC-RFC obtained from agammaglobulinemic and IgA-deficient patients also contained appreciable LT levels even at 1:3 or 15 dilution (Table 4). When compared by Student’s t test to LT levels obtained with EAC-RFC from normal donors, their levels were not significantly (n.s.) different. These results suggested that CRL cells, if appropriately activated by EAC binding to membrane C3 receptors, produce LT irrespective of whether they are from normal individuals or patients with B-cell immunodeficiency syndromes.

DISCUSSION

Previous studies (10, 11) demonstrated that CRL from normal donors activated via membrane C3 receptors nonspecifically destroyed normal and malignant human target cells. EAC and ZC-rosetted non-T cells gave cytotoxic responses which were dependent on neither antibody nor phagocytic cells but involved lymphotoxin. In this paper, the cytotoxic responses of CRL from patients with total or partial B-cell deficiencies were compared to normal CRL. Congenital agammaglobulinemic patients who had neglible numbers of SmIg-staining cells and low circulating immunoglobulin levels provided an opportunity to assess whether the expression of CRL-mediated cytotoxicity was dependent on the presence or capacity to secrete immunoglobulins. CRL from agammaglobulinemic patients gave cytotoxic effector responses statistically comparable to those of CRL from normal donors. The absence of SmIg and serum immunoglobulins in congenital agammaglobuliemic patients did not significantly affect the expression of CRL-mediated cytotoxicity. Furthermore, these results made improbable the explanation that CRL in culture with target cells produced antibodies capable of mediating antibody-dependent cytotoxicity. Our findings with human EAC-RFC are consistent with those of Perlmann et ul. (12) in that CRL rosetted with “‘Cr-labeled EAC did not cause 51Cr release (Mackler, B. F., unpublished data). However, EAC- or ZC-rosetted CRL cultured with a second 51Cr-labeled, proliferating target cell line (e.g., human lung fibroblasts and melanoma cells) gave significant cytotoxic responses. These CRL-mediated cytotoxicity levels are significantly greater than control non-E-RFC (P
CRL

RESPONSES

OF IMMUNODEFICIENT

PATIENTS

287

vious studies (10) in our laboratory obtained quite similar levels of CRL-mediated cytotoxicity with a second target cell line, human lung fibroblasts (WI-38). These results may have reflected target cell heterogeneity in that only a subpopulation was susceptible to cytolysis. CRL-mediated cytolytic responses appeared to be a direct consequence of activation by EAC or ZC binding to C3 membrane receptors since enriched B cells (non-E-RFC) depleted of T cells were not cytotoxic. Non-E-RFC subpopulations repeatedly centrifuged on Ficoll-Hypaque gradients also gave no cytotoxic responses (10). In addition, reconstitution of CRL with E-RFC or N-RFC neither suppressed nor diminished CRL cytotoxicity (Mackler, B. F., and O’Neill, P., unpublished data) suggesting that the expression of CRL-mediated cytotoxicity is not dependent on prior removal of suppressor cells. The. nature of the C3 receptor-bearing cytotoxic cells found in normal and immunodeficient patients remains to be fully established. Previous findings excluded the participation of phagocytic cells (10). The CRL from agammaglobulinemic patients contained appreciably fewer SmIg + cells than similar subpopulations from normal donors. Irrespective of this difference, the CRLmediated cytotoxic responses of the former were statistically similar to those of normals. The majority of SmIg - CRL found in agammaglobulinemic patients may conceivably be an expanded subpopulation of lymphocyte-like cells lacking T or B antigenic markers (13). However, this explanation does not account for the fact that CRL from normal and agammaglobulinemic donors gave almost comparable nonspecific cytotoxic responses. It seems more probable that these CRL are immature B lymphocytes (14) with impaired synthesis of immunoglobulins and surface antigens (13) but still capable of expressing nonspecifically induced effector functions. It is unclear whether one or multiple cytolytic effector mechanisms are expressed by C3-activated non-thymus-derived cells. We were unable to demonstrate CRL-mediated cytotoxicity in the absence of LT in all immunodeficient patients studied. Efforts to separate these human CRL effector mechanisms into discrete subpopulations of different densities have been unsuccessful in contrast to studies with murine T cells (15, 17). These results suggested that LT may be the cytolytic effector mechanism in both direct EAC-RFC and EAC-RFC culture supernatant cytotoxicity assay systems. The lower values in the supematant assays probably reflected the dilutions (1:3 or 1:6) used as compared to the direct EAC-RFC assays. The expression of C3 receptor-induced effector responses does not appear to be dependent on the presence of humoral immunocompetency. This finding is consistent with recent reports that non-thymus-derived cells from patients with similar immunodeficiencies can nonspecifically lyse target cells coated with added antibody (18, 19). In vivo, these nonspecific effector functions are probably not normally expressed because of the lack of sufficient serum antibody or the inability to form immune complexes which bind complement. Thus, in appropriate clinical circumstances the administration of plasma or y-globulin may have an added therapeutic value of enhancing nonspecific host humoral responses mediated by CRL.

288

MACKLER

ET AL.

ACKNOWLEDGMENTS We are grateful to the Clinical Research Center, Texas Childrens Hospital, and staff for their assistance in studying patients. We thank Dr. Philip Wyde for his critical comments and suggestions. Mrs. Dora Woodson, Miss Sharon Edgeworth, and Mrs. Rebecca McCulloch provided excellent technical assistance. This study was supported by USPHS Grants DE-02232, DE-04210, and DE-04172 from the National Institute of Dental Research; General Research Support Grant 5-Sol-RRO5344(13); Clinical Research Center Grant FR-00188; and Grant CA-07357. A postdoctoral fellow (Peggy O’Neill) was supported by NIDR Training Grant I-T22-DE-00035-01.

REFERENCES 1. O’Toole, C., Perlmann, P., Wigzell, H., Unsgaard, B., and Zetterlund, C. G., Lancer 2, 1085, 1975. 2. Lamon, E. W., Skurzak, H. M., Klein, E., and Wigzell, H., J. Exp. Med. 136, 1072, 1972. 3. Mackler, B. F., Altman, L. C., Rosenstreich, D. L., and Oppenheim, J. J., Nuttrre fLondon) 249, 834, 1974. 4. Chess, L.. MacDermott, R. P., and Schlossman, S. F., J. fmmunol. 113, 1113, 1974. 5. Epstein, L. B., Kreth, H. W., and Herzenberg, L. A., Cell Zmmunol. 12, 407. 1974. 6. Mackler, B. F., Altman, L. C., Wahl, S., Rosenstreich, D. L., Oppenheim, J. J.. and Mergenhagen, S. E., Infect. Immun. 10, 844, 1974. 7. Chess, L., MacDermott, R. P., and Schlossman, S. F.. J. Immunol. 113, 1122, 1974. 8. Wahl, S., Iverson, M., and Oppenheim, J. J., J. Exp. Med. 140, 1631, 1974. 9. Altman, L., Chassy, B., and Mackler, B. F., J. Immunol. 115, 18, 1975. 10. Mackler, B. F., and O’Neill, P., Fed. Proc. 34, 1015, 197.5. 11. O’Neill, P., Mackler, B. F., and Wyde, P., Cell. Immunol. 20, 33, 1975. 12. Perlman, P., Perlman, A., and Mtiller-Eberhard. H. G., J. Exp. Med. 141, 287, 1975. 13. Hayward, A. R., and Graves, M. F., Clin. Immunol. Immunopathol. 3, 481. 1975. 14. WHO/IARC Workship, Stand. J. Immunol. 3, 521, 1974. 15. Henney, C. S., Gaffney, J., and Bloom, B. R., J. .Exp. Med. 140, 837, 1974. 16. Tigelaar, R. E., and Gorezynski. R. M., J. Exp. Med. 140, 267, 1974. 17. Dawkins, R. L., and Zilks, P. J., Nature (London) 254, 144, 1975. 18. Rachelefsky, G. S., McConnachie, P. R., Ammann, A. J., Terasaki, P. I., and Stiehm, E. R., Clin. Exp. Immunol. 19, 1, 1975. 19. Froland, S., and Natvig, J. B., Transplant. Rev. 16, 114. 1973.