CELLULAR
IMMUNOLOGY
The Two-Step
37, 209-220
(1978)
Leukocyte
Migration
Its Use in Evaluation
of Cellular
Inhibition Immune
with lmmunodeficiency STANLEY Laboratory
Factor (LIF) Assay
Function
in Patients
Diseases1
W. CHAPMAN 2 AND CHARLES H. KIRKPATRICK
of Clinical Investigation, National Ilrstitutes
National Institute of Allergy of Health, Rethesda, Maryland
Rrceived
October
and Infectious 20014
Diseases,
25, 1977
The in vitro production of leukocyte migration inhibition factor (LIF) by cells derived from normal volunteers and patients with a variety of immunodeficiency diseases has been evaluated. Antigenic specificity was studied with soluble antigens from Can&da albicans and streptokinase-streptodornase (SK-SD) while the mitogens concanavalin-A (Con A) and phytohemagglutinin (PHA) served as non-specific stimuli for LIF synthesis. In normal volunteers, LIF production to antigens was found to be a good ia vitro correlate of delayed cutaneous hypersensitivity. All volunteers also had mitogen-responsive cells. In contrast, certain patients with chronic mucocutaneous candidiasis (CMCC) had an antigen-specific defect when their lymphocytes were stimulated with candida antigen. This defect was associated with active candidiasis and was corrected following successful therapy with amphotericin B and transfer factor. These patients had LIF-producing cells that were responsive to mitogens, and SK-SD-induced LIF production correlated with skin test responses irrespective of the activity of the candidiasis. Patients with more profound immunodeficiencies such as SCID and an anergic patient with disseminated mycobacteriosis did not respond to either mitogenic or antigenic stimulation with LIF production. This correlated with their cutaneous energy, quantitative deficiencies of T-lymphocytes and subnormal lymphocyte transformation to mitogens and antigens. Various in vitro parameters of cellular immunity have been reported as correlates of delayed cutaneous hypersensitivity. Lymphokine production, specifically macrophage migration inhibition factor (MIF) ,3 by lymphocytes stimulated with
specific antigens, has been shown in the guinea pig (1, 2) and man (3) to correlate with skin test responses. Due to technical difficulties with the indirect MIF assay in man, other migration inhibition assays have been developed using human 1 Portions of this work were published in Abstract form in Clinical Research 25, 355A, April, 1977. *Current address: Division of Infectious Diseases, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. a Abbreviations used in this paper: MIF, macrophage migration-inhibition factor ; LIF, leukocyte migration inhibition factor, CMCC, chronic mucocutaneous candidiasis ; SK-SD, streptokinase-streptodornase ; Con A, concanavalin A; PHA, phytohemagglutinin; SCID, severe combined immunodeficiency. 209 000%8749/78/0371-0209$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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leukocytes as the indicator cells. Various technical modifications of the leukocyte migration inhibition factor (LIF) assays have been described using capillary tubes, agarose gels, and one-step or two-step methods (4-7). Experimental data indicate that LIF is distinct from MIF (8), may be an esterase (9) and its synthesis can be stimulated by mitogens as well as antigens (10). However, some authors report problems with reproducibility of the LIF assay and poor correlation with skin test responses (7, ll-13), while others feel that p.articulate antigens are better suited for LIF assays (14, 15). The purpose of this paper is to describe the technique of a two-step assay for LIF in agarose and its application in the in vitro evaluation of cellular immunity in normal volunteers and patients with immunodeficiency diseases. LIF production was found to be a good in vitro correlate of skin test responsiveness in normal volunteers. In contrast, patients with chronic mucocutaneous candidiasis (CMCC) were found to have an antigen-specific defect for Candida albicans which was related to disease activity. The use of a second antigen (SK-SD) and the mitogens Con-A and PHA allowed definition of the specificity of this defect, and mitogens were also useful in evaluation of patients with more severe immunodeficiency states. METHODS
AND
MATERIALS
Subjects. Control blood donors were obtained from the volunteer population at the National Institutes of Health and from healthy laboratory personnel. The clinical features of the patients are summarized in Table 1. There were 14 patients with chronic mucocutaneous candidiasis (CMCC). At the time of their initial NIH evaluations, ten of the patients had negative candida skin tests (five were anergic), but at the time of this investigation some patients had received transfer factor and only five (Cases 5 and 11-14) had negative candida skin tests. The age of onset of candidiasis ranged from 3 months to 63 years. Within this population, six patients also had idiopathic endocrinopathies, one patient had the hyperimmunoglobulin E syndrome, one patient had a malignant thymoma and one infant had severe combined immunodeficiency (SCID). Four patients (Cases 1-4) were evaluated while their disease was inactive following treatment with amphotericin B and transfer factor. One patient (Case 5) was studied while her disease was active and again after therapy, when inactive. The final patient (Case 15) was anergic and had disseminated disease due to a Group III atypical mycobacteria (Battey bacillus) involving the skin, lungs, pleura, peritoneum and bone marrow. Skin testing. Patients and healthy volunteers were skin tested with commercially “O”, 1 : 100, Hollister-Steir Laboraavailable Candida albicans (Dermatophytin tories, Downers Grove, IL) and streptokinase-streptodornase ( (SK-SD) (Varidase, Lederle Laboratories, Pearl River, NY). SK-SD was initially diluted to 40 U SK and 12.5 U SD/ml. If delayed tests with this concentration were negative, they were repeated using a lo-fold greater concentration. Testing involved the intradermal injection of 0.1 ml of antigen, and cutaneous responses were read at 24 and 48 hr. Positive responses produced 0.5 cm or larger of induration. Skin testing was done after the completion of all in vitro studies. Antigens and mitogens for in vitro studies. Antigens used for in vitro studies were prepared from the same commercial antigens used for skin testing. The anti-
LIF
PRODUCTION
IN
IMMUNODEFICIENCY
TABLE
1
Clinical and Immunologic Features of Immunodeficient Patient
1 2 3 4 Sac b 6 7 8 9 10 11 12 13
14 15
211
DISEASE
Patients Disease status
TF therapy
Current age
Age disease onset
Candida albicans skin test
CMCQ CMCC CMCC’ CMCCCMCC
12 years 37 years 12 years 25 years 25 years
18 months 6 years 7 years 9 months 14 years
CMCCb CMCC” CMCG CMCC CMCC? CMCC CMCC CMCC Hyper IgE syndrome CMCC SCID Disseminated Battey Disease
69 years 18 years 14 years 12 years 27 years 9 years 27 years 17 years
63 years 3 years 8 years 3 years 2 years 3 months 6 years 7 years
Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Negative Negative Negative
Inactive Inactive Inactive Inactive Active Inactive Active Active Active Active Active Active Active Active
Yes Yes Yes Yes No Yes No No No Yes No
1 year
3 months
Anergic
Active
NO
Negative
Active Battey Disease
Yes
Diagnosis
22 years
16 years
a Associated Endocrinopathy. b Thymoma. ca, Patient Studied Prior to Transfer Factor Therapy; Factor Therapy.
NO
No NO
b, Patient Studied After Transfer
gens were dialyzed overnight against 0.9% phosphate-buffered saline, reconstituted in medium 199 (GIBCO, Grand Island, NY) to stock solutions and sterilized by filtration through 0.45 pm filters (Millipore, Bedford, MA). Concanavalin A (Miles Laboratories, Elkhart, IN) and phytohemagglutinin (Burroughs Wellcome Co., Research Triangle Park, NC) were also reconstituted in medium 199 and filter sterilized. Stock solutions of antigens and mitogens were prepared so that a 1: 10 dilution in the cell cultures resulted in the desired concentrations. Preparation of agarose Ynedium The agarose medium was prepared according to Clausen (4). In brief, a fresh solution was prepared daily containing 1% agarose (Indubiose A37, Accurate Chemical and Scientific Corp., Hicksville, NY), 10% heat-inactivated horse serum (GIBCO) , single strength medium 199, penicillin (62 III/ml) and streptomycin (80 pg/ml). The pH of the mixture was adjusted with sodium bicarbonate (TC sodium bicarbonate solution, Difco Labs, Detroit, MI) to 7.2-7.4. Six ml of the agarose-serum-medium solution were poured into 60 X 15 mm plastic petri dishes (Falcon Plastics, Oxnard, CA) and allowed to gel. When gel had formed, eight holes were cut using a 2.3 mm stainless steel punch. Generation of LIF-rich supernatants. Lymphocytes were isolated from heparinized blood by gradient centrifugation on ficoll-hypaque (Ficoll-Paque, Pharmacia Fine Chemical, Piscataway, NJ). Cells were then washed three times in Hanks’ balanced salt solution (GIBCO) and resuspended at a concentration of 2 x IO6 lymphocytes per ml in medium 199 modified with Earle’s base (GIBCO), gluta-
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mine, sodium bicarbonate, 10% heat-inactivated pooled human serum (NABI, Miami, FL), penicillin (62 IU/ml) and streptomycin (80 &ml). One ml of the cell suspension was added to upright, round bottom, glass culture tubes, with or without the appropriate concentration of antigen or mitogen. Initial evaluations revealed that optimum antigen concentrations for LIF production by normal cells were 10 or 20 pugof protein per ml for candida and SO/l.25 or 100/25 units/ml for SK-SD. Optimum concentrations of mitogens were 2 ~g/ml for PHA and 40 or 20 pg/ml for Con A. Cultures were incubated for 72 hr at 37°C in a 5% COn water saturated atmosphere. At the end of 72 hr the suspensions were spun at 2000s and the supernatants harvested. The unstimulated culture supernatants were reconstituted with the appropriate concentrations of antigen or mitogen and these served as the controls against which the stimulated supernatants were compared. As immunosuppressive factors have been found in human serum (16) and MIF activity has been found in fetal calf serum (17), each batch of heat-inactivated serum was tested for any effects on antigen and mitogen-induced lymphocyte proliferation, LIF generation and for direct inhibitory or enhancing effects on leucocyte migration. Assay for LIF activity in supernatants. Granulocyte-rich cell suspensions from healthy donors served as the indicators for LIF activity. These cells were obtained from heparinized blood which was first centrifuged at 200g after which the plasma and buffy coat region were aspirated and mixed. The red cells were allowed to spontaneously sediment at 37”C, the granulocyte-rich plasma was aspirated and the cells were washed 3 times in HBSS. The cells were then resuspended at a concentration of 2.2 x lo8 leukocytes per ml in medium 199 with penicillin, streptomycin and 10% pooled human serum. Stained differential counts revealed the percentage of polymorphonuclear leucocytes to range between 52-820/O. Fifty microliters of the granulocyte rich cell suspension was mixed with 200 ~1 of the test supernatants or the reconstituted control supernatants and incubated at 37°C for 90 min. Following incubation the cell concentration was readjusted to 2.2 x 10s/ml and quadruplicate 7 ~1 aliquots were added to the agarose wells and incubated for 18 hr at 37°C in a 2% COZ humid atmosphere. At the completion of incubation 3% glutaraldehyde was added to each dish for 60 min, the agarose was removed, the cells were washed with distilled water and stained with amido black (18). After staining, the plates were washed with distilled water and air dried. The fields of migration were projected on to a screen (Nikon Profile Projector, Japan), traced and measured with a planimeter. The percent of enhancement or inhibition was then calculated using the formula: y0 inhibition
= 1-
mean area migration activated supernatants x 100. mean area migration control supernatants >
Direct or one-step LIF assay. Experiments were also carried out to assess the direct effect of the antigens or mitogens on the indicator cells. The experiments were similar to those described by Clausen (5). Fifty microliters of the indicator cell suspension was mixed with 5 ~1 of antigen or mitogen. Five microliters of media was added to the cells serving as controls. These mixtures were then incubated at 37°C for 90 min, mixed and 7 ~1 aliquots were added in quadruplicate to the agarose wells. Incubation of the plates and subsequent fixation, staining, planimetry reading and calculations were as described previously.
LIF
PRODUCTION
Preparation of dialymble described previously (19).
IN
IMMUNODEFICIENCY
transfer
factor.
213
DISEASE
Transfer
factor
was prepared
as
RESULTS Cha.racteristics of the LIF assays. The initial phase of this investigation were directed toward development of an assay for LIF generation in which soluble antigens from C. albicans and streptococci could be used. Clausen (5) and Rauch and King (20) had published a direct or one-step LIF assay in which the cells that were being tested for lymphokine production and the indicator cells were mixed. However, we were unable to adapt this system to the soluble antigens PPD, SK-SD or candida. Similar difficulties with soluble antigens in the direct assay have been noted by others (11-14). However, this finding was important because it demonstrated that the small number of lymphocytes that contaminated the indicator cell preparations would not respond to antigens in the culture fluids by producing significant amounts of LIF. LIF production by cells from control subjects. Four candida skin test-positive and four candida skin test-negative normal subjects were studied. Each subject was studied on two or three different days to determine variability of the assay. Although there was some day-to-day variation, each skin test-positive subject always produced supernatants that caused at least 15% inhibition of leukocyte migration and no skin test-negative control produced supernatants that inhibited by as much as 15%. These results are summarized in Fig. 1. The mean inhibition for the skin test-positive subjects was 18.76 * 1.1370 and was significantly different from the skin test negative controls (1.25 * l.lOyG inhibition, P < 0.001).
.
i T . :
-3 2. .
-
.
FIG. 1. LIF production by normal subjects. All four can&da-sensitive subjects produced LIF while none of skin test-negative produced significant inhibition. All subjects were skin test-reactive to SK-SD and produced LIF to this antigen. Both Con A and PHA also induced LIF production.
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.
-
. 5:
T
8
.
. .
CANDIDA AlACANS Skin Test Skin Test Positive Negative
”
. -#.
SK-SO Skin Tea Skin Test N@iVV PO.?&
CON A
PHA
FIG. 2. LIF production by patients with chronic mucocutaneous candidiasis. Skin test-negative patients always failed to produce LIF in response to antigenic stimulation. Six of nine candidiasis patients failed to produce LIF in response to candida even though they had positive candida skin tests. All patients produced LIF in response to Con A and PHA.
With cells from normal donors, stimulation with either 10 or 20 pg of candida produced supernatants with essentially equal inhibitory activity. All normal volunteers were skin test-reactive to SK-SD, and each volunteer produced LIF-rich supernatants that were capable of at least 15% inhibition of leukocyte migration (Fig. 1). As with candida, the inhibitory activity of these supernatants was essentially the same at both doses of antigen tested. Also shown in Fig. 1 are the results of LIF generation by cells from normal donors stimulated with Con A or PHA. In general, the degree of inhibition by mitogen-stimulated culture fluids was greater than that produced by antigens. On the basis of these results, LIF production by antigen and mitogen-stimulated cells was considered positive when supernatants inhibited the indicator leukocyte migration by 15% or more. LIF production by cells frow patienfs. The results obtained for patients l-13 are summarized in Fig. 2. All patients were tested on two or more occasions and each point represents the mean for each individual. Six of the ten patients, even though they had positive delayed skin responses to candida, failed to produce supernatants that caused significant inhibition. In this respect, they were indistinguishable from the skin test-negative patients. In contrast, when their cells were stimulated in vitro with SK-SD, six of the nine SK-SD-reactive patients produced supernatants that caused significant inhibition of migration. The mean inhibition by the SK-SD skin test-positive was 18.02 k 3.377 o and was significantly greater than that produced by the skin test-negative patients ( 1.94 + 1.77% ; P < 0.02). Although patient 13 was SK-SD positive, she was excluded from this analysis because her cells consistently responded to SK-SD by producing a factor that markedly enhanced leukocyte migration. It is noteworthy that three SK-SD-positive patients showed dose dependent LIF responses to this antigen. In each case, significant inhibition was noted only when cells were stimulated with the higher dose of SK-SD. In only one patient
LIF
PRODUCTION
IN
IMMUNODEFICIENCY
DISEASE
215
was such a dose effect noted with candida antigen, and as mentioned previously, no such dose dependency was noted in normal volunteers. The results of LIF-production by mitogen stimulation are also shown in Fig. 2. All patients produced LIF when their cells were stimulated by Con A or PHA. In most instances, the degree of leukocyte migration inhibition was greater with mitogen-induced supernatants than with antigens. To further define the relationships between LIF production and skin test reactivity, contingency tables were constructed and analyzed by Fisher’s exact test. These data are presented in Fig. 3a and 3b. No correlation could he made between skin test responses to candida and LIF production by candida-stimulated cells (P > 0.05) (Fig. 3a). I n contrast, skin test reactivity to SK-SD showed a significant (P = 0.047) correlation with LIF production (Fig. 3b). Again patient 13 was not included in this evaluation because of the migration enhancing factor. Patients 14 and 1.5 were analyzed separately because the degree of their immunodeficiency was more profound. In effect they served as negative controls. Case 14 had SCID with deficient numbers and functions of both T and B cells, and his cells showed no LIF production when stimulated with either antigens or mitogens. Patient 15 had a disseminated Battey bacillus infection with chronic leukopenia and deficient lymphocyte transformation to mitogens and antigens. Her cells were also unproductive of LIF after stimulation with either mitogens or antigens. LIF production and disease activity. The relationship between LIF production and activity of mucocutaneous candidiasis was also examined. As shown in Fig. 4, of the five patients with no active disease, three produced significant LIF activity
FIG. 3. Relationships between delayed hypersensitivity responsesand LIF production. (a) Responses to candida showed no correlation between the two responses (P > 0.1). (b) Responsesto SK-SD showed a significant (P = 0.047) correlation between skin responsesand LIF
production
to this
antigen.
216
CHAPMAN
AND
KIRKPATRICK
FIG. 4. Relationship of activity of mucocutaneous candidiasis to LIF production. Note that patients with active candidiasis did not respond to candida with LIF production irrespective of their skin reactivity. In contrast, four of six SK-SD skin test-positive patients responded to this antigen with LIF production, even though they had active candidiasis.
and one was borderline (14.5%). All had positive delayed cutaneous reactions to candida. In contrast, none of the nine patients with active disease produced significant amounts of LIF. The probability that this phenomenon is antigen specific is suggested by the results with SK-SD. Note that two of three skin test positive patients with no active candidiasis and four of six skin test-positive patients with active disease produced significant amounts of LIF (Fig. 4). The relationships of LIF production and skin test responses to disease status are summarized in Fig. 5a and 5b. These contingency analyses showed a highly significant association between failure to produce LIF and active disease (P = 0.027) (Fig. 5a). On the other hand there was no correlation between skin test reactivity and disease status (P = 0.10) (Fig. 5b). Patient 5 was studied both before and after treatment with amphotericin B and transfer factor. Prior to therapy her candida skin test was unreactive, but she had a positive response to SK-SD. At that time LIF production was demonstrable with SK-SD (22.9% inhibition), but not with candida (6.3% inhibition). After therapy her candida skin test became positive and the disease was inactive; her cells now responded to candida by producing LIF (16.0% inhibition). SK-SD stimulated LIF production was unchanged. DISCUSSION This study demonstrates that for soluble antigens from candida and SK-SD, the two-step assay for LIF production provides an Gs vitro correlate of delayed cutaneous hypersensitivity in normal subjects. On every occasion, the skin test positive volunteers yielded supernatants capable of producing at least 15 % inhibition of leucocyte migration. On the contrary, no skin test-negative volunteer produced supernatants with inhibitory activity of this magnitude. Clausen (4) and Weisbart et al. (6) have used a similar two-step assay system for LIF production
LIF
PRODUCTION
IN
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DISEASE
217
STATUS ACTIVE
INACTIVE
I
I
r
DISEASE INACTIVE
DISEASE
STATUS ACTWE
FIG. 5. Relationships between disease activity and immune responses to candida antigens. (a) There was a highly significant association between failure to produce LIF and activity of the candida infection (P = 0.027). (b) In contrast, there was no association between disease activity and cutaneous responses to candida (P = 0.1). Fisher’s exact test.
and have also found good correlation between skin test sensitivity to PPD and LIF production to this antigen. Clausen’s data also indicated that the use of cell free supernatants to assay for LIF production was more sensitive than his one-step or direct method. Since cells from normal donors were used as “indicator cells” in this assay, it was important to determine if there was sufficient contamination of the leukocyte suspensions with lymphocytes to produce lymphokines and cause direct inhibition of cell migration. Three lines of evidence indicate that this did not occur in our system. First, the indicator cells in the direct assay resulted in no inhibition of leukocyte migration from either skin test-positive or skin test-negative controls even though antigens were present. Second, in experiments in which control supernatants were produced but not reconstituted with antigens prior to assay, there were no differences in the areas of cell migration between the antigen-free and antigen-containing supernatants. Finally, antigen-reconstituted control supernatants produced less inhibition than those derived by antigenic stimulation of cells from skin test-reactive donors, A final methodologic point from this study is the importance of using more than one dose of antigen in studies of LIF production by cells from immunedeficient patients. There were four instances in which cells from patients failed to produce significant amounts of LIF with the lower dose of antigens but gave positive responses with the higher dose; this dose dependency was not seen with normal cells. The data also demonstrate that mitogenic stimulation of lymphocytes results in
218
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LIF production that can be assayed with a leucocyte rich indicator cell population. This serves as an antigen-independent assessment of the functional potential of cells to produce lymphokines and can be used as an additional assay in the evaluation of cellular immune function in patients with putative immunodeficiency syndromes. For example, patients 1-13 had cells that were capable of LIF production when stimulated with mitogens even though six of the skin test-positive patients failed to produce LIF after stimulation with candida. The two patients with more severe immunodeficiency syndromes (Cases 14 and 15) failed to produce LIF in response to both antigens and mitogens. Thus, patients with the more common form of chronic mucocutaneous candidiasis have lymphocytes that are potentially capable of synthesizing and secreting lymphokines, but appear to be deficient in specific antigen-responsive cells or to have defects in critical cell interactions (i.e. macrophage-lymphocyte, lymphocyte-lymphocyte) that make them unresponsive to certain antigens. Patients with more severe deficiences such as SCID (Case 14) lack the required lymphocyte populations or have cells that are incapable of responding to any stimuli (Case 15). Induction of LIF with mitogens may also provide a lymphokine reagent or standard as noted by Maini and coworkers (7). Use of a standard preparation enabled them to assess day to day variation in both lymphocyte transformation and lymphokine production by antigen-stimulated lymphocytes. In addition, variations of indicator leucocyte responsiveness could be monitored by including a mitogen-induced supernatant derived from the same patient on a previous day. Our study illustrates that certain patients with chronic mucocutaneous candidiasis have a defect in LIF production that in many cases is selective for antigens from candida. Of particular interest was the finding that the defect was most commonly found in patients with active candidiasis, and was unrelated to patients’ responses to the candida skin test. Therapy with amphotericin B and transfer factor prepared from skin test positive donors was accompanied by correction of the immunologic defect and by clinical benefit. This is illustrated by Case 5, who following treatment became disease free, skin test-positive, and whose lymphocytes responded to candida by producing LIF. The only exception was Case 1, who also became skin test-reactive and disease free after treatment, but who did not produce LIF. This patient later developed extensive cutaneous dermatophytosis due to Trichophyton tonsurans. The specificity of the antigen defect in these patients is illustrated by the findings with SK-SD. SK-SD-stimulated LIF production by the patients correlated with their skin test responses irrespective of the activity of the candidiasis. Of nine patients who did not make LIF in response to candida and had active candidiasis, seven had positive SK-SD skin tests and six of these produced LIF in response to SK-SD; the patient who was skin test positive but LIF negative for SK-SD was case 13 who consistently produced a leucocyte migration enhancing factor. A similar enhancing factor has been described by Weisbart et al. (21) and has been characterized as a lymphokine distinct from LIF. As such, all seven patients could be considered SK-SD-responsive lymphokine producers. Other workers have noted similar antigen specific defects in lymphocyte proliferation and lymphokine production in patients with chronic infectious diseases (22-25). Diamond and Bennett (24), in studying lymphocyte transformation in successfully treated patients with cryptococcosis, noted depressed lymphocyte
LIF
PRODUCTION
IN
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DISEASE
219
stimulation with heat killed cryptococci. The antigen specificity of the defect was supported by the observation that thymidine incorporation by SK-SD-stimulated lymphocytes of the patients was similar to controls. Schimpfi and Bennett (25) and LIF production evaluated skin test responses, lymphocyte transformation in patients after successful therapy for cryptococcosis. They, too found no correlation between skin test responses to cryptococcin and & vitro LIF production to either heat-killed cryptococci or cryptococcin. However, cutaneous sensitivity to SK-SD in the patients did correlate with LIF production and implicated an antigen specific defect. The specificity of this defect was also demonstrated by studies of lymphocyte transformation using the same antigens. Our data differ from theirs in that successful treatment of candidiasis was accompanied by recovery of antigen-dependent LIF generation. This difference is most likely explained by the fact that our patients were treated with both chemotherapy (amphotericin B) and immunotherapy (transfer factor), whereas the cryptococcosis patients received only amphotericin B. The cause of a specific defect in antigen stimulated LIF production cannot be determined from the results of these experiments. Stobo et al. (26) have reported that certain patients with chronic fungal infections and immunologic defects have mononuclear cells with suppressor activities. We have performed similar experiments on the subjects of this study but have not observed an instance in which preculturing the lymphocytes for several days resulted in changes in responsiveness to antigens in the lymphocyte transformation assay. However, experiments in which precultured cells were examined for antigen-induced LIF production have not been done. It should be mentioned that the patients in Stobo’s report developed fungal infections during adulthood and were clinically different from most of the candidiasis patients in our study. Although serum inhibitions have been noted in some patients with CMCC (27), sup ernatant generation in our experiments was done after cells were washed and in the presence of pooled human serum. We have done experiments with both autologous and homologus plasma in lymphocyte proliferation assays and have found no serum inhibitors in these patients. It is well known that in healthy, normal subjects there is a very high level of concordance between cutaneous responses to antigens and the ability of the same antigens to induce thymidine incorporation and lymphokine synthesis by lymphocytes ,in vitro. A reciprocal relationship usually exists in skin test-negative normal subjects. These findings, together with the observation that lymphokine-containing culture fluids produced delayed hypersensitivity-like inflammation following intradermal injection into guinea pigs (28) have led to the postulation that lymphokines are the short range mediators of cellular immune in&unmation. In certain patients with cellular immune deficiency syndromes, an exact association between cutaneous responses to antigens and responses by lymphocytes ifi z&o does not exist. In 1970, Rocklin et al. (29) reported three patients with Hodgkins disease and one patient with rheumatoid arthritis who, in spite of negative skin tests, had positive MIF and Iymphocyte transformation responses to the test antigens. Additional patients with similar disparate responses have been reported by others (25, 30), and several more examples are presented in this paper. In our study, the disparity correlated closely with disease activity. These observations illustrate that the respective roles of the individual lymphokines in delayed type-inflammatory responses have not been defined. We do not
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know which or how many lymphokines are required to produce a positive skin test, or if the important mediators are actually being measured by the currently used assays. Most reports, including our own, describe results with only one lymphokine, and additional studies will be required to establish the relative contributions of the individual lymphokines to production of delayed cutaneous hypersensitivity reactions. ACKNOWLEDGMENT These studies were done in accordance with a research protocol that had been reviewed and approved by the Clinical Research Committee and Clinical Director of the NIAID. The use of SK-SD for skin testing was done with permission from the Food and Drug Administration. The authors are grateful to Dr. David W. Alling for advice on statistical analysis of the data.
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IMMUNOLOGY
The Two-Step
37, 209-220
(1978)
Leukocyte
Migration
Its Use in Evaluation
of Cellular
Inhibition Immune
with lmmunodeficiency STANLEY Laboratory
Factor (LIF) Assay
Function
in Patients
Diseases1
W. CHAPMAN 2 AND CHARLES H. KIRKPATRICK
of Clinical Investigation, National Ilrstitutes
National Institute of Allergy of Health, Rethesda, Maryland
Rrceived
October
and Infectious 20014
Diseases,
25, 1977
The in vitro production of leukocyte migration inhibition factor (LIF) by cells derived from normal volunteers and patients with a variety of immunodeficiency diseases has been evaluated. Antigenic specificity was studied with soluble antigens from Can&da albicans and streptokinase-streptodornase (SK-SD) while the mitogens concanavalin-A (Con A) and phytohemagglutinin (PHA) served as non-specific stimuli for LIF synthesis. In normal volunteers, LIF production to antigens was found to be a good ia vitro correlate of delayed cutaneous hypersensitivity. All volunteers also had mitogen-responsive cells. In contrast, certain patients with chronic mucocutaneous candidiasis (CMCC) had an antigen-specific defect when their lymphocytes were stimulated with candida antigen. This defect was associated with active candidiasis and was corrected following successful therapy with amphotericin B and transfer factor. These patients had LIF-producing cells that were responsive to mitogens, and SK-SD-induced LIF production correlated with skin test responses irrespective of the activity of the candidiasis. Patients with more profound immunodeficiencies such as SCID and an anergic patient with disseminated mycobacteriosis did not respond to either mitogenic or antigenic stimulation with LIF production. This correlated with their cutaneous energy, quantitative deficiencies of T-lymphocytes and subnormal lymphocyte transformation to mitogens and antigens. Various in vitro parameters of cellular immunity have been reported as correlates of delayed cutaneous hypersensitivity. Lymphokine production, specifically macrophage migration inhibition factor (MIF) ,3 by lymphocytes stimulated with
specific antigens, has been shown in the guinea pig (1, 2) and man (3) to correlate with skin test responses. Due to technical difficulties with the indirect MIF assay in man, other migration inhibition assays have been developed using human 1 Portions of this work were published in Abstract form in Clinical Research 25, 355A, April, 1977. *Current address: Division of Infectious Diseases, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. a Abbreviations used in this paper: MIF, macrophage migration-inhibition factor ; LIF, leukocyte migration inhibition factor, CMCC, chronic mucocutaneous candidiasis ; SK-SD, streptokinase-streptodornase ; Con A, concanavalin A; PHA, phytohemagglutinin; SCID, severe combined immunodeficiency. 209 000%8749/78/0371-0209$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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leukocytes as the indicator cells. Various technical modifications of the leukocyte migration inhibition factor (LIF) assays have been described using capillary tubes, agarose gels, and one-step or two-step methods (4-7). Experimental data indicate that LIF is distinct from MIF (8), may be an esterase (9) and its synthesis can be stimulated by mitogens as well as antigens (10). However, some authors report problems with reproducibility of the LIF assay and poor correlation with skin test responses (7, ll-13), while others feel that p.articulate antigens are better suited for LIF assays (14, 15). The purpose of this paper is to describe the technique of a two-step assay for LIF in agarose and its application in the in vitro evaluation of cellular immunity in normal volunteers and patients with immunodeficiency diseases. LIF production was found to be a good in vitro correlate of skin test responsiveness in normal volunteers. In contrast, patients with chronic mucocutaneous candidiasis (CMCC) were found to have an antigen-specific defect for Candida albicans which was related to disease activity. The use of a second antigen (SK-SD) and the mitogens Con-A and PHA allowed definition of the specificity of this defect, and mitogens were also useful in evaluation of patients with more severe immunodeficiency states. METHODS
AND
MATERIALS
Subjects. Control blood donors were obtained from the volunteer population at the National Institutes of Health and from healthy laboratory personnel. The clinical features of the patients are summarized in Table 1. There were 14 patients with chronic mucocutaneous candidiasis (CMCC). At the time of their initial NIH evaluations, ten of the patients had negative candida skin tests (five were anergic), but at the time of this investigation some patients had received transfer factor and only five (Cases 5 and 11-14) had negative candida skin tests. The age of onset of candidiasis ranged from 3 months to 63 years. Within this population, six patients also had idiopathic endocrinopathies, one patient had the hyperimmunoglobulin E syndrome, one patient had a malignant thymoma and one infant had severe combined immunodeficiency (SCID). Four patients (Cases 1-4) were evaluated while their disease was inactive following treatment with amphotericin B and transfer factor. One patient (Case 5) was studied while her disease was active and again after therapy, when inactive. The final patient (Case 15) was anergic and had disseminated disease due to a Group III atypical mycobacteria (Battey bacillus) involving the skin, lungs, pleura, peritoneum and bone marrow. Skin testing. Patients and healthy volunteers were skin tested with commercially “O”, 1 : 100, Hollister-Steir Laboraavailable Candida albicans (Dermatophytin tories, Downers Grove, IL) and streptokinase-streptodornase ( (SK-SD) (Varidase, Lederle Laboratories, Pearl River, NY). SK-SD was initially diluted to 40 U SK and 12.5 U SD/ml. If delayed tests with this concentration were negative, they were repeated using a lo-fold greater concentration. Testing involved the intradermal injection of 0.1 ml of antigen, and cutaneous responses were read at 24 and 48 hr. Positive responses produced 0.5 cm or larger of induration. Skin testing was done after the completion of all in vitro studies. Antigens and mitogens for in vitro studies. Antigens used for in vitro studies were prepared from the same commercial antigens used for skin testing. The anti-
LIF
PRODUCTION
IN
IMMUNODEFICIENCY
TABLE
1
Clinical and Immunologic Features of Immunodeficient Patient
1 2 3 4 Sac b 6 7 8 9 10 11 12 13
14 15
211
DISEASE
Patients Disease status
TF therapy
Current age
Age disease onset
Candida albicans skin test
CMCQ CMCC CMCC’ CMCCCMCC
12 years 37 years 12 years 25 years 25 years
18 months 6 years 7 years 9 months 14 years
CMCCb CMCC” CMCG CMCC CMCC? CMCC CMCC CMCC Hyper IgE syndrome CMCC SCID Disseminated Battey Disease
69 years 18 years 14 years 12 years 27 years 9 years 27 years 17 years
63 years 3 years 8 years 3 years 2 years 3 months 6 years 7 years
Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Negative Negative Negative
Inactive Inactive Inactive Inactive Active Inactive Active Active Active Active Active Active Active Active
Yes Yes Yes Yes No Yes No No No Yes No
1 year
3 months
Anergic
Active
NO
Negative
Active Battey Disease
Yes
Diagnosis
22 years
16 years
a Associated Endocrinopathy. b Thymoma. ca, Patient Studied Prior to Transfer Factor Therapy; Factor Therapy.
NO
No NO
b, Patient Studied After Transfer
gens were dialyzed overnight against 0.9% phosphate-buffered saline, reconstituted in medium 199 (GIBCO, Grand Island, NY) to stock solutions and sterilized by filtration through 0.45 pm filters (Millipore, Bedford, MA). Concanavalin A (Miles Laboratories, Elkhart, IN) and phytohemagglutinin (Burroughs Wellcome Co., Research Triangle Park, NC) were also reconstituted in medium 199 and filter sterilized. Stock solutions of antigens and mitogens were prepared so that a 1: 10 dilution in the cell cultures resulted in the desired concentrations. Preparation of agarose Ynedium The agarose medium was prepared according to Clausen (4). In brief, a fresh solution was prepared daily containing 1% agarose (Indubiose A37, Accurate Chemical and Scientific Corp., Hicksville, NY), 10% heat-inactivated horse serum (GIBCO) , single strength medium 199, penicillin (62 III/ml) and streptomycin (80 pg/ml). The pH of the mixture was adjusted with sodium bicarbonate (TC sodium bicarbonate solution, Difco Labs, Detroit, MI) to 7.2-7.4. Six ml of the agarose-serum-medium solution were poured into 60 X 15 mm plastic petri dishes (Falcon Plastics, Oxnard, CA) and allowed to gel. When gel had formed, eight holes were cut using a 2.3 mm stainless steel punch. Generation of LIF-rich supernatants. Lymphocytes were isolated from heparinized blood by gradient centrifugation on ficoll-hypaque (Ficoll-Paque, Pharmacia Fine Chemical, Piscataway, NJ). Cells were then washed three times in Hanks’ balanced salt solution (GIBCO) and resuspended at a concentration of 2 x IO6 lymphocytes per ml in medium 199 modified with Earle’s base (GIBCO), gluta-
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mine, sodium bicarbonate, 10% heat-inactivated pooled human serum (NABI, Miami, FL), penicillin (62 IU/ml) and streptomycin (80 &ml). One ml of the cell suspension was added to upright, round bottom, glass culture tubes, with or without the appropriate concentration of antigen or mitogen. Initial evaluations revealed that optimum antigen concentrations for LIF production by normal cells were 10 or 20 pugof protein per ml for candida and SO/l.25 or 100/25 units/ml for SK-SD. Optimum concentrations of mitogens were 2 ~g/ml for PHA and 40 or 20 pg/ml for Con A. Cultures were incubated for 72 hr at 37°C in a 5% COn water saturated atmosphere. At the end of 72 hr the suspensions were spun at 2000s and the supernatants harvested. The unstimulated culture supernatants were reconstituted with the appropriate concentrations of antigen or mitogen and these served as the controls against which the stimulated supernatants were compared. As immunosuppressive factors have been found in human serum (16) and MIF activity has been found in fetal calf serum (17), each batch of heat-inactivated serum was tested for any effects on antigen and mitogen-induced lymphocyte proliferation, LIF generation and for direct inhibitory or enhancing effects on leucocyte migration. Assay for LIF activity in supernatants. Granulocyte-rich cell suspensions from healthy donors served as the indicators for LIF activity. These cells were obtained from heparinized blood which was first centrifuged at 200g after which the plasma and buffy coat region were aspirated and mixed. The red cells were allowed to spontaneously sediment at 37”C, the granulocyte-rich plasma was aspirated and the cells were washed 3 times in HBSS. The cells were then resuspended at a concentration of 2.2 x lo8 leukocytes per ml in medium 199 with penicillin, streptomycin and 10% pooled human serum. Stained differential counts revealed the percentage of polymorphonuclear leucocytes to range between 52-820/O. Fifty microliters of the granulocyte rich cell suspension was mixed with 200 ~1 of the test supernatants or the reconstituted control supernatants and incubated at 37°C for 90 min. Following incubation the cell concentration was readjusted to 2.2 x 10s/ml and quadruplicate 7 ~1 aliquots were added to the agarose wells and incubated for 18 hr at 37°C in a 2% COZ humid atmosphere. At the completion of incubation 3% glutaraldehyde was added to each dish for 60 min, the agarose was removed, the cells were washed with distilled water and stained with amido black (18). After staining, the plates were washed with distilled water and air dried. The fields of migration were projected on to a screen (Nikon Profile Projector, Japan), traced and measured with a planimeter. The percent of enhancement or inhibition was then calculated using the formula: y0 inhibition
= 1-
mean area migration activated supernatants x 100. mean area migration control supernatants >
Direct or one-step LIF assay. Experiments were also carried out to assess the direct effect of the antigens or mitogens on the indicator cells. The experiments were similar to those described by Clausen (5). Fifty microliters of the indicator cell suspension was mixed with 5 ~1 of antigen or mitogen. Five microliters of media was added to the cells serving as controls. These mixtures were then incubated at 37°C for 90 min, mixed and 7 ~1 aliquots were added in quadruplicate to the agarose wells. Incubation of the plates and subsequent fixation, staining, planimetry reading and calculations were as described previously.
LIF
PRODUCTION
Preparation of dialymble described previously (19).
IN
IMMUNODEFICIENCY
transfer
factor.
213
DISEASE
Transfer
factor
was prepared
as
RESULTS Cha.racteristics of the LIF assays. The initial phase of this investigation were directed toward development of an assay for LIF generation in which soluble antigens from C. albicans and streptococci could be used. Clausen (5) and Rauch and King (20) had published a direct or one-step LIF assay in which the cells that were being tested for lymphokine production and the indicator cells were mixed. However, we were unable to adapt this system to the soluble antigens PPD, SK-SD or candida. Similar difficulties with soluble antigens in the direct assay have been noted by others (11-14). However, this finding was important because it demonstrated that the small number of lymphocytes that contaminated the indicator cell preparations would not respond to antigens in the culture fluids by producing significant amounts of LIF. LIF production by cells from control subjects. Four candida skin test-positive and four candida skin test-negative normal subjects were studied. Each subject was studied on two or three different days to determine variability of the assay. Although there was some day-to-day variation, each skin test-positive subject always produced supernatants that caused at least 15% inhibition of leukocyte migration and no skin test-negative control produced supernatants that inhibited by as much as 15%. These results are summarized in Fig. 1. The mean inhibition for the skin test-positive subjects was 18.76 * 1.1370 and was significantly different from the skin test negative controls (1.25 * l.lOyG inhibition, P < 0.001).
.
i T . :
-3 2. .
-
.
FIG. 1. LIF production by normal subjects. All four can&da-sensitive subjects produced LIF while none of skin test-negative produced significant inhibition. All subjects were skin test-reactive to SK-SD and produced LIF to this antigen. Both Con A and PHA also induced LIF production.
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.
-
. 5:
T
8
.
. .
CANDIDA AlACANS Skin Test Skin Test Positive Negative
”
. -#.
SK-SO Skin Tea Skin Test N@iVV PO.?&
CON A
PHA
FIG. 2. LIF production by patients with chronic mucocutaneous candidiasis. Skin test-negative patients always failed to produce LIF in response to antigenic stimulation. Six of nine candidiasis patients failed to produce LIF in response to candida even though they had positive candida skin tests. All patients produced LIF in response to Con A and PHA.
With cells from normal donors, stimulation with either 10 or 20 pg of candida produced supernatants with essentially equal inhibitory activity. All normal volunteers were skin test-reactive to SK-SD, and each volunteer produced LIF-rich supernatants that were capable of at least 15% inhibition of leukocyte migration (Fig. 1). As with candida, the inhibitory activity of these supernatants was essentially the same at both doses of antigen tested. Also shown in Fig. 1 are the results of LIF generation by cells from normal donors stimulated with Con A or PHA. In general, the degree of inhibition by mitogen-stimulated culture fluids was greater than that produced by antigens. On the basis of these results, LIF production by antigen and mitogen-stimulated cells was considered positive when supernatants inhibited the indicator leukocyte migration by 15% or more. LIF production by cells frow patienfs. The results obtained for patients l-13 are summarized in Fig. 2. All patients were tested on two or more occasions and each point represents the mean for each individual. Six of the ten patients, even though they had positive delayed skin responses to candida, failed to produce supernatants that caused significant inhibition. In this respect, they were indistinguishable from the skin test-negative patients. In contrast, when their cells were stimulated in vitro with SK-SD, six of the nine SK-SD-reactive patients produced supernatants that caused significant inhibition of migration. The mean inhibition by the SK-SD skin test-positive was 18.02 k 3.377 o and was significantly greater than that produced by the skin test-negative patients ( 1.94 + 1.77% ; P < 0.02). Although patient 13 was SK-SD positive, she was excluded from this analysis because her cells consistently responded to SK-SD by producing a factor that markedly enhanced leukocyte migration. It is noteworthy that three SK-SD-positive patients showed dose dependent LIF responses to this antigen. In each case, significant inhibition was noted only when cells were stimulated with the higher dose of SK-SD. In only one patient
LIF
PRODUCTION
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DISEASE
215
was such a dose effect noted with candida antigen, and as mentioned previously, no such dose dependency was noted in normal volunteers. The results of LIF-production by mitogen stimulation are also shown in Fig. 2. All patients produced LIF when their cells were stimulated by Con A or PHA. In most instances, the degree of leukocyte migration inhibition was greater with mitogen-induced supernatants than with antigens. To further define the relationships between LIF production and skin test reactivity, contingency tables were constructed and analyzed by Fisher’s exact test. These data are presented in Fig. 3a and 3b. No correlation could he made between skin test responses to candida and LIF production by candida-stimulated cells (P > 0.05) (Fig. 3a). I n contrast, skin test reactivity to SK-SD showed a significant (P = 0.047) correlation with LIF production (Fig. 3b). Again patient 13 was not included in this evaluation because of the migration enhancing factor. Patients 14 and 1.5 were analyzed separately because the degree of their immunodeficiency was more profound. In effect they served as negative controls. Case 14 had SCID with deficient numbers and functions of both T and B cells, and his cells showed no LIF production when stimulated with either antigens or mitogens. Patient 15 had a disseminated Battey bacillus infection with chronic leukopenia and deficient lymphocyte transformation to mitogens and antigens. Her cells were also unproductive of LIF after stimulation with either mitogens or antigens. LIF production and disease activity. The relationship between LIF production and activity of mucocutaneous candidiasis was also examined. As shown in Fig. 4, of the five patients with no active disease, three produced significant LIF activity
FIG. 3. Relationships between delayed hypersensitivity responsesand LIF production. (a) Responses to candida showed no correlation between the two responses (P > 0.1). (b) Responsesto SK-SD showed a significant (P = 0.047) correlation between skin responsesand LIF
production
to this
antigen.
216
CHAPMAN
AND
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FIG. 4. Relationship of activity of mucocutaneous candidiasis to LIF production. Note that patients with active candidiasis did not respond to candida with LIF production irrespective of their skin reactivity. In contrast, four of six SK-SD skin test-positive patients responded to this antigen with LIF production, even though they had active candidiasis.
and one was borderline (14.5%). All had positive delayed cutaneous reactions to candida. In contrast, none of the nine patients with active disease produced significant amounts of LIF. The probability that this phenomenon is antigen specific is suggested by the results with SK-SD. Note that two of three skin test positive patients with no active candidiasis and four of six skin test-positive patients with active disease produced significant amounts of LIF (Fig. 4). The relationships of LIF production and skin test responses to disease status are summarized in Fig. 5a and 5b. These contingency analyses showed a highly significant association between failure to produce LIF and active disease (P = 0.027) (Fig. 5a). On the other hand there was no correlation between skin test reactivity and disease status (P = 0.10) (Fig. 5b). Patient 5 was studied both before and after treatment with amphotericin B and transfer factor. Prior to therapy her candida skin test was unreactive, but she had a positive response to SK-SD. At that time LIF production was demonstrable with SK-SD (22.9% inhibition), but not with candida (6.3% inhibition). After therapy her candida skin test became positive and the disease was inactive; her cells now responded to candida by producing LIF (16.0% inhibition). SK-SD stimulated LIF production was unchanged. DISCUSSION This study demonstrates that for soluble antigens from candida and SK-SD, the two-step assay for LIF production provides an Gs vitro correlate of delayed cutaneous hypersensitivity in normal subjects. On every occasion, the skin test positive volunteers yielded supernatants capable of producing at least 15 % inhibition of leucocyte migration. On the contrary, no skin test-negative volunteer produced supernatants with inhibitory activity of this magnitude. Clausen (4) and Weisbart et al. (6) have used a similar two-step assay system for LIF production
LIF
PRODUCTION
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DISEASE
217
STATUS ACTIVE
INACTIVE
I
I
r
DISEASE INACTIVE
DISEASE
STATUS ACTWE
FIG. 5. Relationships between disease activity and immune responses to candida antigens. (a) There was a highly significant association between failure to produce LIF and activity of the candida infection (P = 0.027). (b) In contrast, there was no association between disease activity and cutaneous responses to candida (P = 0.1). Fisher’s exact test.
and have also found good correlation between skin test sensitivity to PPD and LIF production to this antigen. Clausen’s data also indicated that the use of cell free supernatants to assay for LIF production was more sensitive than his one-step or direct method. Since cells from normal donors were used as “indicator cells” in this assay, it was important to determine if there was sufficient contamination of the leukocyte suspensions with lymphocytes to produce lymphokines and cause direct inhibition of cell migration. Three lines of evidence indicate that this did not occur in our system. First, the indicator cells in the direct assay resulted in no inhibition of leukocyte migration from either skin test-positive or skin test-negative controls even though antigens were present. Second, in experiments in which control supernatants were produced but not reconstituted with antigens prior to assay, there were no differences in the areas of cell migration between the antigen-free and antigen-containing supernatants. Finally, antigen-reconstituted control supernatants produced less inhibition than those derived by antigenic stimulation of cells from skin test-reactive donors, A final methodologic point from this study is the importance of using more than one dose of antigen in studies of LIF production by cells from immunedeficient patients. There were four instances in which cells from patients failed to produce significant amounts of LIF with the lower dose of antigens but gave positive responses with the higher dose; this dose dependency was not seen with normal cells. The data also demonstrate that mitogenic stimulation of lymphocytes results in
218
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LIF production that can be assayed with a leucocyte rich indicator cell population. This serves as an antigen-independent assessment of the functional potential of cells to produce lymphokines and can be used as an additional assay in the evaluation of cellular immune function in patients with putative immunodeficiency syndromes. For example, patients 1-13 had cells that were capable of LIF production when stimulated with mitogens even though six of the skin test-positive patients failed to produce LIF after stimulation with candida. The two patients with more severe immunodeficiency syndromes (Cases 14 and 15) failed to produce LIF in response to both antigens and mitogens. Thus, patients with the more common form of chronic mucocutaneous candidiasis have lymphocytes that are potentially capable of synthesizing and secreting lymphokines, but appear to be deficient in specific antigen-responsive cells or to have defects in critical cell interactions (i.e. macrophage-lymphocyte, lymphocyte-lymphocyte) that make them unresponsive to certain antigens. Patients with more severe deficiences such as SCID (Case 14) lack the required lymphocyte populations or have cells that are incapable of responding to any stimuli (Case 15). Induction of LIF with mitogens may also provide a lymphokine reagent or standard as noted by Maini and coworkers (7). Use of a standard preparation enabled them to assess day to day variation in both lymphocyte transformation and lymphokine production by antigen-stimulated lymphocytes. In addition, variations of indicator leucocyte responsiveness could be monitored by including a mitogen-induced supernatant derived from the same patient on a previous day. Our study illustrates that certain patients with chronic mucocutaneous candidiasis have a defect in LIF production that in many cases is selective for antigens from candida. Of particular interest was the finding that the defect was most commonly found in patients with active candidiasis, and was unrelated to patients’ responses to the candida skin test. Therapy with amphotericin B and transfer factor prepared from skin test positive donors was accompanied by correction of the immunologic defect and by clinical benefit. This is illustrated by Case 5, who following treatment became disease free, skin test-positive, and whose lymphocytes responded to candida by producing LIF. The only exception was Case 1, who also became skin test-reactive and disease free after treatment, but who did not produce LIF. This patient later developed extensive cutaneous dermatophytosis due to Trichophyton tonsurans. The specificity of the antigen defect in these patients is illustrated by the findings with SK-SD. SK-SD-stimulated LIF production by the patients correlated with their skin test responses irrespective of the activity of the candidiasis. Of nine patients who did not make LIF in response to candida and had active candidiasis, seven had positive SK-SD skin tests and six of these produced LIF in response to SK-SD; the patient who was skin test positive but LIF negative for SK-SD was case 13 who consistently produced a leucocyte migration enhancing factor. A similar enhancing factor has been described by Weisbart et al. (21) and has been characterized as a lymphokine distinct from LIF. As such, all seven patients could be considered SK-SD-responsive lymphokine producers. Other workers have noted similar antigen specific defects in lymphocyte proliferation and lymphokine production in patients with chronic infectious diseases (22-25). Diamond and Bennett (24), in studying lymphocyte transformation in successfully treated patients with cryptococcosis, noted depressed lymphocyte
LIF
PRODUCTION
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DISEASE
219
stimulation with heat killed cryptococci. The antigen specificity of the defect was supported by the observation that thymidine incorporation by SK-SD-stimulated lymphocytes of the patients was similar to controls. Schimpfi and Bennett (25) and LIF production evaluated skin test responses, lymphocyte transformation in patients after successful therapy for cryptococcosis. They, too found no correlation between skin test responses to cryptococcin and & vitro LIF production to either heat-killed cryptococci or cryptococcin. However, cutaneous sensitivity to SK-SD in the patients did correlate with LIF production and implicated an antigen specific defect. The specificity of this defect was also demonstrated by studies of lymphocyte transformation using the same antigens. Our data differ from theirs in that successful treatment of candidiasis was accompanied by recovery of antigen-dependent LIF generation. This difference is most likely explained by the fact that our patients were treated with both chemotherapy (amphotericin B) and immunotherapy (transfer factor), whereas the cryptococcosis patients received only amphotericin B. The cause of a specific defect in antigen stimulated LIF production cannot be determined from the results of these experiments. Stobo et al. (26) have reported that certain patients with chronic fungal infections and immunologic defects have mononuclear cells with suppressor activities. We have performed similar experiments on the subjects of this study but have not observed an instance in which preculturing the lymphocytes for several days resulted in changes in responsiveness to antigens in the lymphocyte transformation assay. However, experiments in which precultured cells were examined for antigen-induced LIF production have not been done. It should be mentioned that the patients in Stobo’s report developed fungal infections during adulthood and were clinically different from most of the candidiasis patients in our study. Although serum inhibitions have been noted in some patients with CMCC (27), sup ernatant generation in our experiments was done after cells were washed and in the presence of pooled human serum. We have done experiments with both autologous and homologus plasma in lymphocyte proliferation assays and have found no serum inhibitors in these patients. It is well known that in healthy, normal subjects there is a very high level of concordance between cutaneous responses to antigens and the ability of the same antigens to induce thymidine incorporation and lymphokine synthesis by lymphocytes ,in vitro. A reciprocal relationship usually exists in skin test-negative normal subjects. These findings, together with the observation that lymphokine-containing culture fluids produced delayed hypersensitivity-like inflammation following intradermal injection into guinea pigs (28) have led to the postulation that lymphokines are the short range mediators of cellular immune in&unmation. In certain patients with cellular immune deficiency syndromes, an exact association between cutaneous responses to antigens and responses by lymphocytes ifi z&o does not exist. In 1970, Rocklin et al. (29) reported three patients with Hodgkins disease and one patient with rheumatoid arthritis who, in spite of negative skin tests, had positive MIF and Iymphocyte transformation responses to the test antigens. Additional patients with similar disparate responses have been reported by others (25, 30), and several more examples are presented in this paper. In our study, the disparity correlated closely with disease activity. These observations illustrate that the respective roles of the individual lymphokines in delayed type-inflammatory responses have not been defined. We do not
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know which or how many lymphokines are required to produce a positive skin test, or if the important mediators are actually being measured by the currently used assays. Most reports, including our own, describe results with only one lymphokine, and additional studies will be required to establish the relative contributions of the individual lymphokines to production of delayed cutaneous hypersensitivity reactions. ACKNOWLEDGMENT These studies were done in accordance with a research protocol that had been reviewed and approved by the Clinical Research Committee and Clinical Director of the NIAID. The use of SK-SD for skin testing was done with permission from the Food and Drug Administration. The authors are grateful to Dr. David W. Alling for advice on statistical analysis of the data.
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