Clin. exp. Immunol. (1992) 89, 351-355
Elevated serum levels of soluble tumour necrosis factor receptors (sTNF-R) in patients with HIV infection A. KALINKOVICH, H. ENGELMANN*, N. HARPAZ, R. BURSTEIN, V. BARAKt, I. KALICKMANt, D. WALLACH* & Z. BENTWICH R. Ben-Ari Institute of Clinical Immunology, Kaplan Hospital, Hebrew University Medical School, *Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, and tOncology Department, Hadassah University Hospital, Jerusalem, Israel
(Acceptedfor publication 22 May 1992)
SUMMARY Serum levels of the soluble form of tumour necrosis factor receptor type II (p75) (sTNF-R) were determined in HIV-infected individuals and risk groups and were then correlated with the course of infection and prognosis. sTNF-R levels were determined by an ELISA with MoAbs and polyclonal antibodies to urine-derived sTNF-R proteins. The mean + s.e. levels of sTNF-R in the sera of 49 HIV+ male homosexuals, 34 HIV- male homosexuals and 44 matched controls were 61 + 03 ng/ml, 4.4 + 0 3 ng/ml and 3 4 + 0 2 ng/ml, respectively. All these values were significantly different between each of the groups (P < 0-001-0 05). Sequential studies of sTNF-R revealed higher levels following seroconversion in 5/8 individuals, remained persistently high during the asymptomatic phase of the infection and became even more elevated in some ARC and AIDS patients. At the same time TNF-X was undetectable in sera obtained from HIV+ male homosexuals and from healthy controls. This was independent of stage of HIV infection, serum sTNF-R level and type of ELISA kit used. These findings suggest that TNF-ot/TNF-R system is turned on before and during HIV infection and raise the possibility that sTNF-R, the natural inhibitor of TNF, may be of importance in determining the course and probably prognosis of the disease. Keywords soluble tumour necrosis factor receptors tumour necrosis factor male homosexuals HIV infection
INTRODUCTION S and A of mitdyeral roleforTNFinthepourtse:o inftond hasceent been implicated by several reports:HiV (i) blood monocytes AIDS paiet AIDS atiets poduc spotaneosly ihlvl igh evel offTFc NF-a . [1-3] and react more sensitively to lipopolysaccharide (LPS) stimulation by increased synthesis in vitro [3,4]; (ii) TNF. facilitates the transcription, replication and expression of HIV . macroin human T lymphocytes, different',T cell lines and in phages, and exerts cytocidal influence on them [5-9]; (iii) multiple effects of TNF-oc such as lipoprotein lipase suppression, pyrogenic activity, disturbed CNS function, and inflammation' . all account ' for several of the promoting properties, could clinical features of AIDS, such as wasting, fever, dementia and host susceptibility to several opportunistic infections [10-12]; (iv) TNF-a is able to counteract the anti-HIV activity of azidothymidine and other dideoxynucleosides in vitro [13]. Recently, urine and sera of healthy, febrile and cancer patients has been found to contain two proteins (p55 and p75)
~ prdc.pnaeul ~ TNF-.
which specifically bind TNF-ac. They are of an approximate molecular weight of 30 kD each and provide protection against the cytocidal effect of TNF-ax in vitro. Based several lines eiec hyaeteslbefrso h w onoeua pceof evidence they are the soluble forms of the two molecular species of the cell surface TNF receptors (sTNF-R) and function as physiological inhibitors of TNF-ax activity [14-22]. It was also found that sera of some patients with sarcoidosis, tuberculosis disease cause inhibition of ~~~~~and cytotoxic of TNF-ax aciCrohn's probabl reflectinhebpresn suchTNF-Rin
athe
[23]. them [23]. These observations have led us to determine serum sTNF-R inlevels HIV-infected ~~~~~~~~~levels oupatientsHIV-infected m and risk groups. The markedly indviduals andeis or to tourse ofe disease, . prompted this report.
PATIENTS AND METHODS Most patients were part of a male homosexual cohort that is being followed up at the Kaplan Medical Centre since 1983. They are seen and examined at regular intervals, and their clinical and immune status are evaluated regularly.
Correspondence: Professor Z. Bentwich, R. Ben-Ani Institute of Clinical Immunology, Kaplan Hospital, Hebrew University Medical School, Rehovot 76100, Israel.
351
352
A. Kalinkovich et al. Table 1. Serum levels of sTNF-RII and TNF-a in different patient groups
Diagnosis
sTNF-RII, ng/ml median (range)
Asymptomatic* (2)t AIDS* (5)
AIDSt (4) Lymphoma* (4) Breast carcinomat (3) Bone marrow transplantationt Osteoporosist (1) Chronic renal failure* (4) Normal* (14) Normal* (5)
HIV-infected 5-8 (5-3-6-3) 7 4 (6 4-7 8) 8-1 (7-2-10 2) Other diseases (TNF-a+ controls) ND ND (4) ND ND 6 3 (5-8-7-6) Normal controls ND 3-6 (1-0-5 2)
TNF-a, pg/ml median (range) 2-1 (0-4-2) 1 3 (0-4 4) 0 (0-1-6) 320 (82-573) 50 (28-98) 291 (78-580) 673 190 (140-300)
0-1 (0-14) 0-5 (0-12)
* Fresh sera. t Numbers in parentheses are the number of patients. : Freeze-thawed sera. ND, Not done.
HIV-1 antibody sero status was determined by ELISA and confirmed by Western blot assays. Patients were classified according to the Walter Reed (WR) staging system criteria [24]. Thirty-nine individuals presented with WR stages 1-4 (considered as asymptomatic) and 10 were WR stages 5-6 (ARC/ AIDS). The controls were 44 age- and sex-matched healthy normal (non-homosexual) blood donors. For the study of serum TNF-a, additional patients with chronic lymphocytic leukaemia (n=2), hairy cell leukaemia (n=2), breast carcinoma (n=4), chronic renal failure (n=4), osteoporosis (n = 1) and recipients of bone marrow transplants (n=4) were included. Patients with these diseases have high serum levels of TNF-a [25-29]. Evaluation of sTNF-R and TNF-ac levels was carried out on stored serum samples frozen at -70°C immediately after separation. Most serum samples were thawed only once before testing ('fresh sera') and some were freeze-thawed two-to-three times. Determination of sTNF-R level was performed as described [30]. Briefly, MoAbs specific to the sTNF-R type II [31] were adsorbed to 96-well ELISA plates (Corning, NY). After 2 h incubation at 37°C with a solution of 25 yg/ml ofthe antibody in PBS, the wells were incubated with blocking solution (PBS, 1% bovine serum albumin (BSA), 0-02% NaN3 and 0 05% Tween 20). Tested samples were applied in aliquots of 80 pl/well. The plates were incubated for 2 h at 37°C, thoroughly washed and the rabbit polyclonal antiserum to the sTNF-R type II [18], diluted 1: 500 in blocking solution, was added to the wells. After further overnight incubation at 4°C, the plates were rinsed again and incubated for 2 h with horseradish peroxidase-conjugated purified goat anti-rabbit IgG (BioMakor, Israel). The assay was developed using 2,2'-azino-bis-(3-ethylbenzthiazoline-6 sulphonic acid) (Sigma) as a substrate. The enzymatic product was determined colorimetrically at 405 nm. Purified urine-derived sTNF-R type II [18] served as a standard. The detection limit of the assay was 30 pg/ml. The readings were not affected by repeated freezing and thawing of the sera (see Table 1). In view of the evidence that type II TNF-a receptor is the prevalent
species in cells of the haematopoietic system affected by HIV [18,20], we focused all our studies on this type. Tests of sTNF-R type I (p55) are now ongoing. Serum levels of TNF-cx were assayed using two ELISA kits: (i) Factor.m hTNF-x ELISA Test Kit (Genzyme Corporation, Cambridge, MA) able to detect TNF-a levels greater than 12 pg/ ml; and (ii) Biokine TNF Test Kit (T Cell Sciences, Cambridge, MA) able to detect TNF-a levels greater than 10 pg/ml. Determinations were performed according to the manufacturer's instructions.
RESULTS The results of serum sTNF-R determination in the various groups studied are depicted in Fig. 1. In 49 HIV+ male 20
Is
16_ 14
12 0
6~~~~~~~~~~~~~~~~ 6-
6 1 + 0.3 ; (2490)
*gsg
4.4+ 4 *44+ 0.3 34+02 t °
o
66
00
*ontlrtobts IFig. 1. sTNF-R serum levels in HIV- and HIV+ male homosexuals. Each point represents one individual. The horizontal lines and numbers represent the mean values + s.e. The differences between groups are significant (P
Elevated serum levels of soluble tumour necrosis factor receptors (sTNF-R) in patients with HIV infection A. KALINKOVICH, H. ENGELMANN*, N. HARPAZ, R. BURSTEIN, V. BARAKt, I. KALICKMANt, D. WALLACH* & Z. BENTWICH R. Ben-Ari Institute of Clinical Immunology, Kaplan Hospital, Hebrew University Medical School, *Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, and tOncology Department, Hadassah University Hospital, Jerusalem, Israel
(Acceptedfor publication 22 May 1992)
SUMMARY Serum levels of the soluble form of tumour necrosis factor receptor type II (p75) (sTNF-R) were determined in HIV-infected individuals and risk groups and were then correlated with the course of infection and prognosis. sTNF-R levels were determined by an ELISA with MoAbs and polyclonal antibodies to urine-derived sTNF-R proteins. The mean + s.e. levels of sTNF-R in the sera of 49 HIV+ male homosexuals, 34 HIV- male homosexuals and 44 matched controls were 61 + 03 ng/ml, 4.4 + 0 3 ng/ml and 3 4 + 0 2 ng/ml, respectively. All these values were significantly different between each of the groups (P < 0-001-0 05). Sequential studies of sTNF-R revealed higher levels following seroconversion in 5/8 individuals, remained persistently high during the asymptomatic phase of the infection and became even more elevated in some ARC and AIDS patients. At the same time TNF-X was undetectable in sera obtained from HIV+ male homosexuals and from healthy controls. This was independent of stage of HIV infection, serum sTNF-R level and type of ELISA kit used. These findings suggest that TNF-ot/TNF-R system is turned on before and during HIV infection and raise the possibility that sTNF-R, the natural inhibitor of TNF, may be of importance in determining the course and probably prognosis of the disease. Keywords soluble tumour necrosis factor receptors tumour necrosis factor male homosexuals HIV infection
INTRODUCTION S and A of mitdyeral roleforTNFinthepourtse:o inftond hasceent been implicated by several reports:HiV (i) blood monocytes AIDS paiet AIDS atiets poduc spotaneosly ihlvl igh evel offTFc NF-a . [1-3] and react more sensitively to lipopolysaccharide (LPS) stimulation by increased synthesis in vitro [3,4]; (ii) TNF. facilitates the transcription, replication and expression of HIV . macroin human T lymphocytes, different',T cell lines and in phages, and exerts cytocidal influence on them [5-9]; (iii) multiple effects of TNF-oc such as lipoprotein lipase suppression, pyrogenic activity, disturbed CNS function, and inflammation' . all account ' for several of the promoting properties, could clinical features of AIDS, such as wasting, fever, dementia and host susceptibility to several opportunistic infections [10-12]; (iv) TNF-a is able to counteract the anti-HIV activity of azidothymidine and other dideoxynucleosides in vitro [13]. Recently, urine and sera of healthy, febrile and cancer patients has been found to contain two proteins (p55 and p75)
~ prdc.pnaeul ~ TNF-.
which specifically bind TNF-ac. They are of an approximate molecular weight of 30 kD each and provide protection against the cytocidal effect of TNF-ax in vitro. Based several lines eiec hyaeteslbefrso h w onoeua pceof evidence they are the soluble forms of the two molecular species of the cell surface TNF receptors (sTNF-R) and function as physiological inhibitors of TNF-ax activity [14-22]. It was also found that sera of some patients with sarcoidosis, tuberculosis disease cause inhibition of ~~~~~and cytotoxic of TNF-ax aciCrohn's probabl reflectinhebpresn suchTNF-Rin
athe
[23]. them [23]. These observations have led us to determine serum sTNF-R inlevels HIV-infected ~~~~~~~~~levels oupatientsHIV-infected m and risk groups. The markedly indviduals andeis or to tourse ofe disease, . prompted this report.
PATIENTS AND METHODS Most patients were part of a male homosexual cohort that is being followed up at the Kaplan Medical Centre since 1983. They are seen and examined at regular intervals, and their clinical and immune status are evaluated regularly.
Correspondence: Professor Z. Bentwich, R. Ben-Ani Institute of Clinical Immunology, Kaplan Hospital, Hebrew University Medical School, Rehovot 76100, Israel.
351
352
A. Kalinkovich et al. Table 1. Serum levels of sTNF-RII and TNF-a in different patient groups
Diagnosis
sTNF-RII, ng/ml median (range)
Asymptomatic* (2)t AIDS* (5)
AIDSt (4) Lymphoma* (4) Breast carcinomat (3) Bone marrow transplantationt Osteoporosist (1) Chronic renal failure* (4) Normal* (14) Normal* (5)
HIV-infected 5-8 (5-3-6-3) 7 4 (6 4-7 8) 8-1 (7-2-10 2) Other diseases (TNF-a+ controls) ND ND (4) ND ND 6 3 (5-8-7-6) Normal controls ND 3-6 (1-0-5 2)
TNF-a, pg/ml median (range) 2-1 (0-4-2) 1 3 (0-4 4) 0 (0-1-6) 320 (82-573) 50 (28-98) 291 (78-580) 673 190 (140-300)
0-1 (0-14) 0-5 (0-12)
* Fresh sera. t Numbers in parentheses are the number of patients. : Freeze-thawed sera. ND, Not done.
HIV-1 antibody sero status was determined by ELISA and confirmed by Western blot assays. Patients were classified according to the Walter Reed (WR) staging system criteria [24]. Thirty-nine individuals presented with WR stages 1-4 (considered as asymptomatic) and 10 were WR stages 5-6 (ARC/ AIDS). The controls were 44 age- and sex-matched healthy normal (non-homosexual) blood donors. For the study of serum TNF-a, additional patients with chronic lymphocytic leukaemia (n=2), hairy cell leukaemia (n=2), breast carcinoma (n=4), chronic renal failure (n=4), osteoporosis (n = 1) and recipients of bone marrow transplants (n=4) were included. Patients with these diseases have high serum levels of TNF-a [25-29]. Evaluation of sTNF-R and TNF-ac levels was carried out on stored serum samples frozen at -70°C immediately after separation. Most serum samples were thawed only once before testing ('fresh sera') and some were freeze-thawed two-to-three times. Determination of sTNF-R level was performed as described [30]. Briefly, MoAbs specific to the sTNF-R type II [31] were adsorbed to 96-well ELISA plates (Corning, NY). After 2 h incubation at 37°C with a solution of 25 yg/ml ofthe antibody in PBS, the wells were incubated with blocking solution (PBS, 1% bovine serum albumin (BSA), 0-02% NaN3 and 0 05% Tween 20). Tested samples were applied in aliquots of 80 pl/well. The plates were incubated for 2 h at 37°C, thoroughly washed and the rabbit polyclonal antiserum to the sTNF-R type II [18], diluted 1: 500 in blocking solution, was added to the wells. After further overnight incubation at 4°C, the plates were rinsed again and incubated for 2 h with horseradish peroxidase-conjugated purified goat anti-rabbit IgG (BioMakor, Israel). The assay was developed using 2,2'-azino-bis-(3-ethylbenzthiazoline-6 sulphonic acid) (Sigma) as a substrate. The enzymatic product was determined colorimetrically at 405 nm. Purified urine-derived sTNF-R type II [18] served as a standard. The detection limit of the assay was 30 pg/ml. The readings were not affected by repeated freezing and thawing of the sera (see Table 1). In view of the evidence that type II TNF-a receptor is the prevalent
species in cells of the haematopoietic system affected by HIV [18,20], we focused all our studies on this type. Tests of sTNF-R type I (p55) are now ongoing. Serum levels of TNF-cx were assayed using two ELISA kits: (i) Factor.m hTNF-x ELISA Test Kit (Genzyme Corporation, Cambridge, MA) able to detect TNF-a levels greater than 12 pg/ ml; and (ii) Biokine TNF Test Kit (T Cell Sciences, Cambridge, MA) able to detect TNF-a levels greater than 10 pg/ml. Determinations were performed according to the manufacturer's instructions.
RESULTS The results of serum sTNF-R determination in the various groups studied are depicted in Fig. 1. In 49 HIV+ male 20
Is
16_ 14
12 0
6~~~~~~~~~~~~~~~~ 6-
6 1 + 0.3 ; (2490)
*gsg
4.4+ 4 *44+ 0.3 34+02 t °
o
66
00
*ontlrtobts IFig. 1. sTNF-R serum levels in HIV- and HIV+ male homosexuals. Each point represents one individual. The horizontal lines and numbers represent the mean values + s.e. The differences between groups are significant (P
