Tumor necrosis factor in familial mediterranean fever

Tumor Necrosis Factor in Familial Mediterranean Fever AMI SCHATTNER, M.D., MOTI LACHMI, M.D., Rehovot, Israel, AVI LIVNEH, M.D., MORDECHAI PRAS, M.D., TelHashomer, Israel, TALIA HAHN, Ph.D., Rehovot, lsrael

PURPOSE: The pleiotropic inflammatory effects of tumor necrosis factor (TNF) prompted a study of this cytokine in familial Mediterranean fever (FMF), a recurrent polyserositis of unknown etiology. PATIENTSANDMETHODS: Thirty-sixasymptomatic and 24 patients with acute FMF were studied and compared with 20 matched healthy subjects. TNF levels were measured by bioassay in the plasma and in supernatants of peripheral blood mononuclear cells (PBMC) incubated alone or with an inducer (lipopolysaccharide, phytohemagglutinin [PHA], or Sendai virus). Cytotoxicity could be abolished in all cases by preincubation with monoclonal anti-TNF-a antibodies. RESULTS: No TNF was found in plasma and non-induced PBMC superuatants. Induced TNF production was markedly decreased in patients with acute FMF and increased in asymptomatic FMF patients to levels over those of control subjects (p CO.05). Thus, PI&induced TNF levels were 4 U/mL in patients with acute FMF, 25 U/ mL in asymptomatic patients, and 14 U/mL in healthy control subjects (median values), and the other inducers gave similar results. Retesting of patients first studied during an acute episode when their disease was quiescent also revealed a fivefold increase in TNF production. These results were independent of the use of colchicine, which also had no effect on TNF levels when taken by volunteers (1 mg/day) or when added to the PBMC cultures (low7 M). CONCLUSIONS: Since TNF has a very short half-life in plasma, the capacity of PBMC to respond to TNF inducers may more accurately reflect its synthesis. A marked decrease in thii response in acute FMF suggests “exhaustion” of

cells that are already highly activated to produce TNF and the possible participation of TNF in the pathogenesis of FMF.

F

amilial Mediterranean fever (FMF) is an autosomal recessive disorder occurring most commonly in Sephardic Jews and Armenians [l]. Two phenotypic features characterize the disease: brief, episodic, febrile episodes of peritonitis, pleuritis, or synovitis beginning in childhood or adolescence; and the development of type A amyloidosis, which is manifested clinically mainly by a nephropathy [2]. The pathogenesis of FMF remains obscure, but its typical acute transient intense inflammatory response has prompted us to examine the possibility that an uncontrolled release of inflammatory mediators, in particular the cytokine tumor necrosis factor (TNF), may be involved in the pathogenesis. TNF (also termed cachectin) is a polypeptide hormone with pleiotropic activities that may be either beneficial or deleterious to the host according to the circumstances and amount of its production [3,4]. TNF is well established as a primary mediator in the pathogenesis of septic shock [5] and very possibly cachexia [6], but its role as a putative mediator of the inflammatory reaction is no less intriguing. TNF acts as a pyrogen, both by a direct hypothalamic effect and by inducing the production of interleukin-1 (IL-l) [7]. It activates neutrophils and affects their adherence, infiltration, and accumulation, as well as their phagocytic and cytotoxic activities [8]. In addition, TNF has multiple activating effects on endothelium that increase leukocyte adhesion, vascular permeability, and the expression of major histocompatibility complex antigens of both classes [g-11]. It is also a potent inducer of acutephase reactants [12] and of many other important mediators of inflammation such as IL-l, interleukin-6, prostaglandins, and leukotrienes [3]. The large amounts of TNF that can be produced by stimulated macrophages (up to 2% of their total protein biosynthesis) [4,13] may also contribute to its significant role in mediating the inflammatory reaction. To assess a possible function of TNF in the pathogenesis of the poorly understood recurrent inflammation of-FMF, we have examined TNF pro-

From the Department of Medicine “A” (AS, ML) and the Pediatric Research Center (TH), Kaplan Hospital, Rehovot, Israel, and the Department of Medicine “F” (AL, MP), Sheba Medical Center, Tel Hashomer, Israel. Requests for reprints should be addressed to A. Schattner, M.D., Department of Medicine “A”. Kaplan Hospital, Rehovot 76100, Israel. Manuscript submitted March 15, 1990. and accepted in revised form November 19, 1990.

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duction in vivo and in vitro in patients with active and inactive disease who regularly attend our FMF clinic.

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Spontaneous and Induced Production of TNF PBMC were obtained from heparinized blood by

ET AL

TABLEI Characterization of the Patient Population and Control Subjects

PATIENTS AND METHODS Patients More than 1,000 FMF patients are followed at the Heller Institute for Medical Research, Sheba Medical Center, Tel Hashomer [2]. We have randomly selected 60 patients fulfilling the diagnostic criteria for FMF as previously described [14], who attended the clinic and who gave their informed consent for the study. A detailed history and clinical evaluation were performed, and blood was drawn for routine laboratory tests, determination of acute-phase reactants, which included C-reactive protein and serum amyloid A, and for separation of peripheral blood mononuclear cells (PBMC) and TNF studies as indicated below. Most patients were young (mean age: 29.5 years) Sephardic Jews of North African origin who were taking colchicine 1 to 2 mgl day. About two thirds of the patients had a positive family history of FMF. The patient population characteristics are shown in Table I. Patients with evidence of amyloidosis were excluded from the study. Twenty-four of the patients presented with a typical history and physical findings of an acute episode of FMF. Eleven of them were newly diagnosed cases and therefore untreated or had omitted to take their medication. The clinical diagnosis of acute FMF was supported in all cases by the presence of at least three of the following criteria: (1) elevated erythrocyte sedimentation rate (40 mm/ hour or greater, Westergren); (2) elevated white blood cell count (greater than 10,000/mm3); (3) increased levels of C-reactive protein (greater than 6 mg/mL); (4) increased levels of serum amyloid A (greater than 1,000 units). Seven of the patients first examined during an acute episode were restudied 4 to 20 weeks later while entirely asymptomatic. In addition, samples of synovial fluid aspirated from acutely inflamed joints of FMF patients and stored at -70°C were available for study. Twenty healthy age- and sex-matched volunteers of Sephardic origin served as control subjects, and four of them were examined before and after treatment with colchicine 1 mg/day for several days. Colchitine (E. Lilly & Co., Indianapolis, Indiana; 10m7 M) was also added to PBMC cultures of several healthy controls at different time points (-3 hours, 0 hours, or +3 hours) of the addition of an inducer, and the effect on TNF production was measured as indicated below.

FEVER / SCHATTNER

Acute Men (number of cases) Women (number of cases) Mean age (years) Sephardic origin (%) Family history of FMF (%) Total cases

FMF Asymptomatic

Healthy Controls

16 8

25 11

13 7

;i 62.5 24

;7

1;;

iit

0

separation over Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) and suspended at a concentration of 5 X lo6 cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics (RPMI-FCS). Aliquots of 100 FL were incubated at 37°C for 5 hours in medium alone or in the presence of one of the following inducers: phytohemagglutinin (PHA, Wellcome Diagnostics, Temple Hill, Dartford, United Kingdom), 20 yg/mL; bacterial lipopolysaccharide extracted from Escherichia coli (LPS, Makor Chemicals, Jerusalem, Israel), 10 bg/mL; or Sendai virus 500 HA/mL (gift of Dr. Y. Yohas). Quantitation of TNF Activity Cytotoxicity was quantitated as described [15]. Briefly, 24 hours after seeding HeLa cells in g-mm microwells at 5 X lo4 cells/well, cell-free PBMC supernatants or plasma samples were applied to the cells in serial dilutions in the presence of 40 pg/mL cycloheximide. Sixteen hours later, cell death was quantitated by measuring the uptake of neutral red vital dye, using a Micro-ELISA autoreader (SLT Lab Instruments, Austria) at 540 pm. One TNF unit was defined as the concentration at which 50% of the target cells were killed. This was calculated by plotting a curve of optical density as a function of supernatant concentration. An internal standard was included in each TNF bioassay. All TNF assays on patient and control material were performed in balanced groups. Identification of Cytotoxicity as TNF To confirm that cytotoxicity was due to the production of TNF, neutralization of cytotoxic samples was performed using a monoclonal anti-TNF-cr antibody (gift of BASF/Knoll, Ludwigshafen, Federal Republic of Germany). A total of 200 ng/mL of the monoclonal antibody (1 mg protein/ml) neutralizes 1 ng/mL TNF-LY. Each sample was incubated for 24 hours at 4’C with and without the antiTNF antibody, then retested for cytotoxicity by bioassay as described. An excess of antibody (20 Fg/ mL) was used in all cases. The antibody preparation alone was not cytotoxic for the target cells. April

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TABLEII InducedTNFProductionby PBMCof Patientswith FMF* Inducer+

LPS

PHA

Healthy controls (n = 20) Asymptomatic FMF(n = 36) Acute FMV (n = 24)

39 f 45 (21) 55 f 45.5 (30) 23 f 28 (12)

14.5 f 6 (14) 37 f 36.5 (25) 12.5 i 25 (4)

46.0 f 56 (23.0) 76.5zk 51.5(63.5) 32.5 f 36 (23.5)

Healthy versus asyrrjptomatic Healthy versus acute Asymptomatic versus acute

p <0.05 NS p <0.005

p <0.005 p
p zol p CO.005

Sendai Virus

NS = not significant. The Wilcoxon two-sample rank test was performed using the Clinstat computer program. * Mean f SD (median) is given in U/mL of TNF measured by bioassay as described, following a short-term incubation of patients’ PBMC with the inducer. t With RPM1 alone (no inducer), no SpontaneousTNF production could be found in the majority of patients with FMF. (Zero mean and zero median TNFfor healthy, asymptomatic FMF, and acute FMF alike.) Three asymptomatic patients, however, showed some TNF production (mean levels of 11 U/mL). * No significant differences were discerned between patients who were taking colchicine (n = 11) and the others.

TABLEIll LPS-Induced TNF Productionin Vitroby PBMCof FMFPatients EvaluatedDuringan AcuteEpisodeand WhileAsymptomatic LPS-Induced Acute FMF

Patient : i : 7

TNF (U/mL)* Asymptomatic

Ratio7

32 28

143 173

z 47

2 220

i:; 5.2 4.7

3:

1::

CO

Determined as described in “Quantitation t Median ratio is 5.0.

of TNFActivity”

4.5

in Patients and Methods

Investigation of the Mechanism of Altered TNF Production in FMF

To study the mechanism of altered TNF production in FMF, PBMC of two healthy controls and two asymptomatic patients with FMF were first incubated as described above for 12 hours in RPMIFCS alone or in the presence of TNF (5 ng/mL) or LPS (10 rg/mL). Spontaneous TNF production, as well as the response to TNF inducers (LPS or PHA), was then assessed as described above, after washing the cells three times with RPMI. Cell viability was not significantly affected. Statistics

Since the distribution of the data in each of the groups was clearly not normal, a non-parametric test was used, namely, the Wilcoxon two-sample test, using the Clinstat computer program. RESULTS None of the patients or control subjects had detectable TNF in their serum. Spontaneous TNF production by PBMC incubated with medium alone was also very rare and encountered in very few asymptomatic patients and in none of the other groups (Table II). All synovial fluids examined (n = 6) were negative for TNF. Induced TNF production, however, was signifi436

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cantly reduced in the symptomatic patients as compared with the group with inactive FMF (p <0.005), regardless of whether the inducer used was LPS, PHA, or Sendai virus. The latter was the most potent TNF inducer followed by LPS, but PHA was the more sensitive inducer and gave the best discrimination between the various groups (Table II). Thus, median PHA-induced TNF production in acute FMF was 4 U/mL as compared with 14 U/mL in healthy controls and 25 U/mL in asymptomatic FMF. The validity of this observation of low levels of induced TN.F during acute FMF, which was significant regardless of whether the patient was receiving colchicine or not (not shown), is greatly enhanced by our re-evaluation of the same patients when their disease was quiescent (Table III). All patients who were re-examined showed about a fivefold increase in LPS-induced TNF production compared with their own levels during acute FMF. This finding was entirely in line with the levels observed in the whole group of 36 asymptomatic patients, which were even significantly higher than those of the healthy controls (by 1.5 to 2.5-fold) (Table II). When healthy volunteers were tested before and after colchicine treatment, no consistent effect of the drug on TNF production could be discerned (not shown), and in uitro incubation of control PBMC with colchicine at different time points in relation to the addition of the inducer likewise had no effect on TNF production. Finally, to study whether the decreased response to TNF inducers in acute FMF could be due to feedback inhibition by high levels of TNF itself or to exhaustion of the cells following their response to postulated TNF inducers in &JO, we examined the response of PBMC to challenge with TNF inducers following treatment in vitro with TNF or LPS. The results are shown in Table IV. We found that prior TNF exposure had no significant effect on the response to inducers in controls and asymptomatic FMF patients alike. However, while control PBMC exposed to LPS could still produce TNF at levels

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comparable with PBMC incubated alone, we could demonstrate that PBMC from FMF patients to LPS considerably depressed TNF response neously and when challenged by either (Table IV).

with RPM1 exposure of resulted in a both spontaLPS or PHA

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TABLE IV TNF Production by PBMC from Healthy Control Subjects and Asymptomatic FMF Patients Following Pretreatment for 12 Hours with TNF (5 ng/mL) or LPS (10 pg/mL) in CriW

Pretreatment

COMMENTS This study examines the possible participation of TNF, a cytokine with prominent inflammatory effects, in the pathogenesis of the acute episode of FMF, which is still poorly understood. Our main finding is that in patients with acute disease, induced production of TNF by PBMC in vitro is severely depressed, regardless of the inducer used. When these “active” patients are compared with a group of asymptomatic FMF patients, the difference is especially striking (p <0.005) since induced TNF levels were found to be elevated in asymptomatic patients, who produced significantly more TNF than healthy control subjects (p X0.05). The validity of these observations is further augmented by the study of patients with acute FMF who were reexamined several weeks later while entirely asymptomatic and who showed a fivefold increase in induced TNF levels in remission. These results indicate that significant changes in TNF may occur during the course of FMF. The fact that colchicine, which is remarkably effective in preventing most episodes of FMF [1,2], had no effect on TNF in our studies may either indicate that our findings are incidental to the pathogenesis or that the drug is active more distally, perhaps on mediators and changes induced by TNF that are important in inflammation [16,17]. The lack of demonstrable serum or spontaneous TNF production in our patients does not negate our conclusion. First, TNF has a very short plasma halflife (6 minutes) and may have already been cleared from the plasma or bound to its receptors [3,18]. Second, TNF bioactivity may be observed in very low serum levels, which may even be undetectable [19,20]. Finally, the time lapse of 2 to 5 hours between venipuncture and processing of the blood may have also had an adverse effect on the possibility of detecting TNF in the circulation. However, it is just because of such problems that the measurement of the capacity of PBMC in uitro to respond to inducing agents has become an accepted alternative, leading to several important observations in diverse conditions [21-251. The significance of our finding of decreased TNF inducibility in acute FMF is presently unclear. However, it may reflect the presence of cells that had been already activated to produce TNF and therefore could not amply respond to further stimulation. Our interpretation

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None TNF LPs*

Inducer of TNF Productiont None LPS PHA Control FMF Control FMF Control FMF

6 2

8

iz

2

0

25

;;

:

5

ii

5

8

* Results are the mean in two closely matched individuals. f Numbers denote U/mL of TNF. r An assay of the supernatants of PEMC at 12 hours revealed TNF levels in the range of 100 to 150 U/mL.

is supported by the results of the experiments summarized in Table IV, which do not support feedback inhibition by high levels of TNF itself as the explanation for our findings in acute FMF, but rather suggest that PBMC of FMF patients respond poorly to a TNF inducer following a previous induction of TNF, and are quite different in that respect from PBMC of healthy controls. This type of phenomenon has already been reported for interferon in systemic lupus erythematosus (SLE) [26] and myasthenia gravis [27], for IL-l in acquired immunodeficiency syndrome (AIDS) [28], and for TNF itself in a mouse model of murine SLE 1291. High levels of TNF from exogenous sources have also been associated with depressed in vitro responses to a TNF inducer [30]. The opposite observation, namely, that of increased induced TNF synthesis in asymptomatic FMF, may be related to the same process and reveal a possible “compensatory” increase in TNF production capacity secondary to previous episodes of activation. Alternatively, other factors may also explain the depressed in vitro response to TNF inducers in acute FMF. These include changes in other cytokines that may either suppress or enhance the response to TNF inducers as recently demonstrated in other systems [31,32]. The possible presence of soluble-specific cell surface receptors for TNF must also be considered [33], since these may also be present in FMF and interfere with TNF bioactivity. These results should be further pursued to achieve a better understanding of the changes in TNF during the course of FMF and its possible contribution to the pathogenesis and the diagnosis of FMF.

ACKNOWLEDGMENT We are grateful to Dr. Meir Azur, Department of Mathematics, ty, for his valuable help in the statistical analysis.

Tel-Aviv Universi-

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