Humoral and cellular immunity in the anergic tuberculosis patient

Humoral and cellular immunity in the anergic tuberculosis patient A prospective

study

Stanley

J. Zeitz,

M.D.,

Arsdel,

Jr., M.D.

Sctrfflc,

Jonathan I\7cr.sh.

H. Ostrow,

M.D.,

and

Paul

P. Van

VOLUME 53 NUMBER 1

Immunity

in anergic

tuberculosis

21

opened, lasted about 30 days. One person administered and then determined all the PPD test reactions, which were recorded in millimeters of induration at 48 hours. Care was taken to avoid repeat testing at the same skin site. Disposable plastic tuberculin syringes were used. Reactions of 5 mm. or less were considered negative. Negative reactors were promptly retested with second-strength PPD (250 TU) and the results again interpreted in 48 hours. Patients who were anergic to second-strength PPD were accepted for further study, and additional medical diagnoses, medications received, and other personal or family history suggesting immunologic deficiency were also determined. The appearance of the tonsils, size and consistency of lymph nodes, and temperature ranges at the time of skin testing were noted. Routine laboratory studies, including the total and differential blood cell count, were obtained, and the patients received further skin tests with histoplasmin, coccidioidin, mumps, and Candida antigens. Additional special studies included Schick tests, typhoid anti-O- and anti-H antibodies before and after typhoid vaccination, red blood cell isohemagglutinins, and quantitative serum immunoglobulins G, A, and M. Serum was also obtained for determiThe sera were tested by a standard nation of precipitating antibodies to tuberculoprotein. micro Ouehterlony technique. Best results were obtained by placing undiluted serum in the center well and twofold dilutions of PPD, (Parke, Davis & Co.) varying from 15.6 to 250 pg per milliliter, in the peripheral wells. Precipitin lines appeared in all positive tests within 3 days, though all slides were observed for a week before being discarded. of cell-rich plasma Cell-mediated immunity was further studied in vitro. One milliliter was added to 4 ml. of TC 199 culture medium and incubated at 37” C. Cultures with nothing added were compared with 3 day cultures containing phytohemagglutinin j&o (Difco Laboratories) and 6 day cultures containing 5 fig per milliliter PPD. Higher concentrations of PPD tend to be toxic to cells and were not studied. Lymphocyte activity was measured by the uptake of radioactivity by the cultured lymphocytes 2 hours after a pulse dose of tritiated thymidine was added (5 pCi per 5~ml. of culture). The anergic patients were then retested monthly with PPD-S until a positive (greater than 9 mm.) reaction occurred; and the lymphocyte tissue culture studies were then repeated, as were the serologic studies. For comparison, similar studies were performed on 41 patients with positive PPD skin reactions and active disease and 7 PPD-positive employees with inactive disease. RESULTS

Of 458 patients with active tuberculosis confirmed by positive sputum cultures,* 6 (1.34 per cent) were nonreactive to both PPD-S and second-strength PPD. One of these was an 83-year-old woman with generalized lymphosarcoma and minimal active pulmonary tuberculosis. Shortly after the skin testing she was treated with chlorambucil and prednisone, in addition to isoniazid and ethambutol, but she died a few weeks later. The remaining 5 patients were all Caucasian males ranging in age from 41 to 89 years, each of whom had advanced cavitary pulmonary tuberculosis with sputum positive on both smear and culture (Table I). None of these 5 patients had a history suggesting possible cause for immunologic deficiency other than the tuberculosis itself, which was clearly active but neither miliary nor overwhelming. None of the patients had significant lymphadenopathy or tonsillomegaly. All were febrile at the time of the initial skin testing, with, temperatures ranging from 100° to 104.6O F. Only 1 patient had definite peripheral lymphopenia. His lymphocyte count gradually rose toward normal during treatment. The remainder were within the usual range *All organisms were specifically identified as Mycobaoteriwm Firland Hospital laboratory, which is the reference acid-fast Washington.

tuberculosis laboratory

hominis in the for the state of

22

Zeitz,

Ostrow,

TABLE I. Anergic

Patient

1 Age

D.G.G. R.H.B.

82 41 57 X9

CA1C.C.

C.W. G.F.P. F.J.F.

7%

83

TABLE II. Anergic

and

patients,

1 Race

C. (‘. (‘. (‘. (‘. (‘.

/

J. ALLERGY CLIN. IMMUNOL. JANUARY 1974

Van Arsdel

Firland

Stax

1

Hospital,

Extent

1969-1971

of

M

Pulmonary:

Advanced

E M M F

Pulmonary

: Noncavitary

patients-laboratory

Additional associated

tuberculosis

studies

Now Xone Now Nonr Sonr Lymphosarooma

eavitary

on admission

D.G.G. H.H.B.

7,650 14,600

22 9

1,680 1,310

Neg. Nrg.

(‘.Mc.C.

12,750

:i

3x5

PO%

ST”

9.200

17

1.560

ST”

Kane

9,750

13

1,270

Pas.

C.W. G.F.P. *ST:

diagnoses with anergy

None 0 + 1: 80

0 -+

1:160

Anti-B 1 :X Anti-A and li I:128 Anti-A 1 : 16 Anti-R l-32 ., Anti-A 1 : 16 Anti-B 1: 16 ST*

not tested.

TABLE III. Serum immunoglobulins Patient D.G.G.

K.H.H. C.Mc.C. C.W. G.F.P. Controls

Sample 1

2 1 2 I 2 1 2 1 2 1 2

in anergic

patients* W

1,700 1,200 3,300 1,600 1,025 1,100 1,440 1,850 2,750 1,850 1,717 1.477

ISM

W

245 130 350 270 425 280 600 520 205 300 498 364

*Sample 1 was obtained at the time of admission and Sample 2, after of treatment. Controls were 15 patients with active tuberrulosis (mean va.lues are given).

1,850 1,050 162 94 103 94 60 51 6X 125 138 93 approximately and positive

4 months skin tests

for Firland Hospital patients and did not change significantly. Two of 4 patients tested had positive Schick tests, and i! of 1 failed to show a rise in anti-O- 01 anti-H antibodies after typhoid vaccination (Table II). Eone of the 5 developed positive skin reactions to mumps, coccidioidin, histoplasmin, or Candida. All patients were treated with isoniazid and rthambutol in standard doses, and 4 of the 5 also received streptomycin. None of the 5 received para-aminosalicylic acid, an agent that might have weak anti-inflammatory actions. Each patient made an uneventful clinical recovery from his tuberculosis, all becoming culturenegative within 6 months of beginning antituberculosis chemotherapy. When retested monthly with PPD-S, all showed eventual return of skin test positivity.

Immunity

VOLUME 53 NUMBER 1 TABLE

IV. Uptake

of tritiated

Patients

/

Positive PPD with Inactive TB Positive PPD with Active Tn

N

thymidine

by lymphocytes

Cells alone

1

/

Initial

365*

Repeat 1 nitial

322 360

in anergic

tuberculosis

in culture

Cells + PHA 66,088"

Celis + PPD

/ 12,545"

7

p < 0.20 127,272 48,518

40,649 1,463

15

p < 0.10 After treatment

Yegativr PPD with Active TR

23

Initial D.G.G. R.H.B. C.McC. C.W. G.F.P. Mean

338

51,428

47

18

49 168 289

21,397

611

708 4,203 5,157

138

5,449

5,766

256 63 159 249 470 206 \

After

treatment

Mean D.G.G. R.H.B. C.McC. C.W. G.F.P.

256

61,049

1,801

30 301

3,185 75,691

181 1,831

16,487

1,987

84,838 20,388

487 3,243

109 140 301

p<

0.0004

/

*Counts per 10 minutes.

ranging from 59 to 134 days from the date of original (average 93.8 days). Serologic

studies

and

lymphocyte

tissue

culture

negative

skin tests

results

Except for 2 patients with high IgG levels and 1 with a high IgM level, there was no major difference in serum immunoglobulins between anergic patients and those whose tuberculin tests were initially positive. Generally, the high immunoglobulin levels were present initially and dropped as the disease came under control (Table III). Preeipitins to PPD were found in the sera of all 5 anergic patients. These developed best at antigen concentrations between 31 and 125 pg per milliliter and in all cases were single, discrete lines. Sera collected later, after positive PPD skin responses had developed, were tested at the same time as the pretreatment sera. Three of the 5 had become negative. In contrast only 3 out of 41 patients with active disease and initial positive PPD responses had precipitating antibodies to PPD. In vitro cell-mediated immunity was compared in 3 groups of donors. The first group was 7 employees of the tuberculosis hospital who had inactive disease but a positive PPD response and who had not been tested with PPD for several years. Their lymphocytes showed a very brisk response to phytohemagglutinin and also to PPD in culture (Table IV). The 7 employees were then skin-tested and the positive reaction to PPD confirmed. Three to 6 weeks later their lymphocyte response in culture was again tested. The change in uptake of radioact.ivity was not statistically significant. The second group consisted of patients

24

Zeitz,

Ostrow,

and Van Arsdel

J. ALLERGY CLIN. IMMUNOL. JANUARY 1974

with active tuberculosis ant1 positive PPT) skin tests. Lymphocytes from this group showed a good response to phytohemagglutinin but ronsidcrably less to PPJ). Four to 6 months later lymphocytes from these same patients hat1 an increased response to PI’I) that was of hordcrline significance. The lymphocytes from the third group, consisting of the 5 ancrgie patients with active tuberculosis, had a ponsiderahly diminished rcsponst~ to $1)~tohemagglutinin ant1 did nol rcspon(l at all to adtlctl 1’1’1). Onccx the ant>rgic: patients’ tuberculin skin tests beca.mc positive, their lymphocyte rcqxtnsr to phytohemagglutinin increasctl and the response to 1’1’1) approximated the initial activity. of the lymphocytes of the tuberculosis patients in group 2 (Table 1V 1. The rise in lymphocyte response to phytohcmagglutinin and tuberculin in the ancrgic patients following treatment was highly significant (p < 0.000-l). The on(patient (l>.(:.tr.) whose cell rcsponsc to WI) (lid not increase was also one of the, two whose sera continued t,o show precipitins. In a preliminary study uv found that I ml. of serum from an anergic patient (G.F.P.) blocked the PPT> stimulation in culture of washed lymphoc+ytes from a proved responder, but, the (*ells respondetl as ctxpectcd in the presence of autologous serum or other Pl’I)-positive sera. Washed anergic lymphocytes did not respond to 1’1’1) in the wbscncc of strum nor could these same cells bc mad<, responsive in the presener either ot’ st’rum from a good rcspontlcr or thtl supernatant, solution from a responding culture following 17’1) stimulation. DISCUSSION

Tuberculin anergy may occur secondary to two distinct classes of clinical states. The first is associated with diseases or treatment known to suppress the expression of cellular immunity, such as Hodgkin’s disease and related reticuloentlotlielial malignancies,‘-” other solid fumors,’ sarcoidosis,” treatment wit,h corticosteroids,“~ 7 treatment with lymphocytoxic agents,“, 9 malnutrition,“’ coexisting infection with certain viruses, or administration of vaccines.“, ” The second includes critically ill patients, whether the underlying disease be tuberculosis itself’” or other unrelated acute conditions” where the immunocyte may he already totally committed or diminished, as in the very old patient.l”T “’ After the various “secondary” causes of tuberculin hyporeactivity have been excluded, the possibility of faulty test material or technique must he wJnSideret1. Common problems include improper dilution of tuberculin solutions, bacterial (Gontamination, inactivation due to exposure to excessive heat or light, and atisorption of tuherculoprotein to walls of vials and syringes.” This latter proI)lcm can largely be eliminated by addition of a stabilizing detergent, such as twcen 80, to the PIW solution.” Another problem has been the wide disparit! of bioactivity noted between some commercial I’Pl)‘s that were generally weight rather than hioassayed for tuberculoprotein content.‘g More recently, standardized tuberculoprotein solutions, which are both bioassayed and tween 80stabilized, have become available; and currently all commercial I’PJ) products conform to this sfandard2” In the present study the PPD-S used for screening was bioassayed but did not, contain tween 80. Thus, some of the smaller tuberculin reactions may have been due to injection of subpotent doses because

VOLUME 53 NUMBER 1

Immunity

in anergic

tuberculosis

25

of adsorption of some of the tuberculoprotein to the walls of the glass containers and plastic tuberculin syringes. However, the use of a much larger (250 TU) second-strength test dose, to which the anergic patients likewise failed to react, clearly shows that a major impairment of delayed hypersensitivity was presentwhich cannot be attributed to subpotent tuberculin dose. The excellent correlation between the in vitro lymphocyte responsiveness and tuberculin skin reactivity rules out the possibility that the negative skin tests were simply due to observer error. Once faulty t.est materials, faulty techniques, and “secondary” causes of anergy have been excluded, there remains a group of patients (not as rare as once thought) who have tuberculin unresponsiveness without obvious cause. The frequency of anergy has been estimated as 0.3% to 0.6% of all patients with active pulmonary tuberculosis, based on retrospective analysis of hospitalized tuberculosis patients. 2*-23In the present prospective study, energy was present in 6 (1.3%) of the 458 tuberculosis patients. Only 1 of these could be explained by any of the factors discussed above. In the other 5, the anergic state disappeared within 3 months after treatment was started and would have been missed if skin tests had been performed only during their convalescence. Since patients who have active tuberculosis and positive PPD skin tests also showed less of a lymphocyte response to PPD than those with inactive tuberculosis, and since normal responsiveness was restored with treatment, it may be that anergy could be the extreme end of a tendency to deficient cellmediated immunity associated with active tuberculosis. All the anergic patients in the present study initially had demonstrable precipitins, whereas only 3 of the 41 control (tuberculin-positive) tuberculosis patients had circulating precipitins. It. is also noteworthy that only 2 of the 4 patients had positive Schick tests, and 2 of the 4 failed to respond with serologic responses to typhoid vaccine. Additionally, 1 of the patients had low red cell isohemagglutinin titers, indicating that a variable degree of nonspecific deficiency of other aspects of humoral immunity may accompany humoral hyperimmunitp to PPD. Were cellular anergy restricted to PPD, one could speculate that humoral antibody bound the PPD and prevented effective contact with lymphocytes. However, this is not the case. Possibly an associated factor temporarily interferes with substances such as the mitogenic factor and other lymphokines that are necessary for the expression of the cellular immune response.24 This possibility is supported by our observation that serum from an anergic patient inhibited the response of known sensitive lymphocytes to PPD in culture and similar observations made in other diseases (reviewed by Dwyer and Kantor25). However, the mechanism for the T cell responses being specifically inhibited (PPD) as well as diffusely impaired (other antigens and PHA) remains unclear. What is clear is tha.t depression of cell-mediated immunity and lymphocyte responsiveness was not restricted to PPD in these patients. We are grateful to Miss Elizabeth Amdall, Miss Miriam Gray, and Mrs. Vinita their technical assistance and for the statistical analysis by Mr. Jack H. Taub.

Cady for

26

Zeitz,

Ostrow,

and Van Arsdel

J. ALLERGY CLIN. IMMUNOL. JANUARY 1974

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