Immunobiol., vol. 181, pp. 317-323 (1990)

1 National

Eye Institute, and 2Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, U.S.A.

Serum Neopterin Levels Following Intravenous Endotoxin Administration to Normal Humans JEFFREY N. BLOOM 1, ANTHONY F. SUFFREDINI2 , JOSEPH E. P ARRILL0 2 , and ALAN C. PALESTINE 1 Received February 15, 1990 . Accepted in Revised Form June 18, 1990

Abstract Endotoxin was administered intravenously to five normal subjects. Measurement of serum neopterin levels demonstrated no significant change from baseline during the first 6 h after endotoxin administration, but were elevated two to four-fold at 24 h. In the three subjects in whom it was measured, a two-fold rise of the mean serum neopterin levels persisted at 48 h. The acute inflammatory events initiated by endotoxin administration to normal humans result in a delayed, but sustained, rise in serum neopterin levels which persists well after the acute phase response has subsided.

Introduction Neopterin is a low molecular weight pteridine compound which is produced as a metabolite in the biosynthetic pathway from guanosine triphosphate to tetrahydrobiopterin (1, 2). It has been reported to be secreted exclusively by macrophages in response to stimulation by interferon-y, produced from activated T-Iymphocytes (1). Although its function in the immune response is unknown (2), neopterin has been utilized as an indicator of cellular immune activation (3, 4). Increased levels of serum and urine neopterin have been noted in several disorders, including the acquired immune deficiency syndrome (5, 6), rheumatoid arthritis (7), tuberculosis (8), ulcerative colitis (9), Crohn's disease (10), coeliac disease (11), graft versus-host disease (12), multiple sclerosis (13), sarcoidosis (14), acute anterior uveitis (15), and various malignancies (16). Recently, it has been suggested that neopterin levels may be useful for the evaluation of the clinical course of patients with sepsis (17). We report here the results of our investigation of the serum neopterin levels of normal humans following the administration of intravenous endotoxin. Materials and Methods Four men and one woman (ages 27 to 39 years of age, mean 30 years) were studied. The current investigation was performed simultaneously with an evaluation of the effects of

318 .

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BLOOM, A. F. SUFFREDINI, J. E. PARRILLO, and A. C. PALESTINE

endotoxin on pulmonary function. The preliminary results of this portion of the investigation have been detailed elsewhere (18). The study was approved by the Clinical Research Subpanel of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. Written consent was obtained from each of the test subjects after the nature and the potential risks of the protocol were explained to them. After fasting for eight h, the subjects were admitted to the medical intensive care unit and remained at bed rest and fasting for six to eight h. Following local infiltration with 1 % lidocaine, radial artery catheters (20 gauge) were placed percutaneously to continuously monitor blood pressure and to provide access for blood sampling. Intravenous fluids were administered at maintenance levels through a peripheral intravenous catheter. U.S. Standard Reference Endotoxin (4 ng/kg, Lot EC-5, U.S. Food and Drug Administration, Bethesda, MD, USA) was administered intravenously over 1 to 2 min after baseline vital signs and blood samples were obtained. This dose of endotoxin has been shown in previous studies to have a characteristic dose-response relationship for the resulting febrile response and alterations in peripheral leucocyte counts (19). Blood pressure, pulse, respiratory rate and oral temperature were monitored hourly following endotoxin administration. Blood samples for total and differential leucocyte counts were obtained at 0, 0.5, 1, 1.5,2, 4, 6, and 24 h following endotoxin. The bioactivity of tumor necrosis factor was measured with a modification of an assay for cell cytotoxicity using a sensitive mouse fibrosarcoma cell line (WEHI 164 clone 13, kindly supplied by Dr. T. ESPEVICK (20, 21) as previously described in detail (22). Serum samples for tumor necrosis factor determinations were obtained at 0, 1, 1.5, 3, 5, and 24 h post-endotoxin. Serum neopterin levels were measured at 0, 1,3,6, and 24 h following endotoxin administration using a commercially available radioimmunoassay (Neopterin - RIAcid/Serum, Henning, Berlin, West Germany). Serum neopterin levels were additionally obtained at 48 hand 72 h from three subjects; and at one week after endotoxin administration in one subject. Ten ml of blood were collected in serum separator glass tubes covered with aluminum foil to prevent exposure to ambient light, and allowed to clot at room temperature. The serum was isolated by centrifugation, within 1 h of the blood collection, and was transferred to aluminum foilcovered vials and stored at -20°C. Descriptive statistics are expressed as mean and standard error of the mean. Statistical analysis was performed with the Statistical Analysis System (SAS, Cary, North Carolina, USA) using the Duncan multiple-range test with an a = 0.001 and the Wilcoxon signed rank test.

Results All of the subjects developed symptoms of chills, myalgias, headache, and mild nausea at one h after the endotoxin infusion. These symptoms were most prominent at 2 to 3 h and began to abate 3 to 4 h following the endotoxin administration. By 24 h after endotoxin, the subjects were completely asymptomatic. Clinical and laboratory measurements are summarized in Table 1. Oral temperature rose from a baseline of 36.44 ± 0.10 °C to a maximum of 38.38 ± 0.22 °C after 3 to 4 h (p < 0.05). The heart rate rose from a mean of 63 ± 5 to 106 ± 4 beats/min (p < 0.05) and mean arterial pressure fell from 83 ± 5 to 71 ± 1 mm Hg (p < 0.05). Total leucocyte counts fell from 4.4 ± 0.3 x 109 to 2.2 ± 0.4 x 109 cells/L at 1 h and then steadily rose to 9.8 ± 1.2 x 109 cells/L at 6 h and remained elevated at 24h (9.9 ± 1.2 x 109 cells/L, p < 0.05). The activity of tumor necrosis factor was undetectable at baseline in all subjects. Maximum levels of tumor necrosis factor were detected at 1 to 1.5

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113 95 119 101 104

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24 h

Peripheral Blood Leukocytes (xl0 9 cells/L)

Table 1. Clinical measurements in five normal humans following intravenous endotoxin administration

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post-endotoxin (500 ± 246 pg/mL), and approached baseline levels by 3 h (25 ± 6 pg/mL). Tumor necrosis factor was undetectable at 5 hand 24 h post-endotoxin. Serum neopterin levels did not change significantly from baseline during the first 6 h following endotoxin administration. At 24 h, serum neopterin levels rose significantly, two to four-fold above baseline values. A two-fold elevation of mean serum neopterin levels persisted in the three subjects evaluated at 48 h (Fig. 1). Elevation of serum neopterin levels were moderately associated with maximum tumor necrosis factor levels. No strong association was noted between the fall in mean arterial pressure and tumor necrosis factor or serum neopterin.

Discussion Endotoxin is believed by many investigators to playa central role in the pathogenesis of gram-negative septic shock. Studies based on endotoxin administration to normal humans using identical preparations and doses have shown that endotoxin initiates multiple arms of the acute-phase response. These include fever, a hyperdynamic cardiovascular state, the release of stress hormones, activation of fibrinolysis, neutrophil activation, as well as the release of endogenous inflammatory mediators, including interleukin-l ~ and tumor necrosis factor (22-26). The initial acute phase responses, including the detection of tumor necrosis factor, occur primarily within the first 3 h following endotoxin administration (23-26). The current study shows that neopterin, an indirect marker of T -lymphocyte activation of macrophages, has a delayed rise for at least six h and then increases two to four-fold by 24 h following intravenous endotoxin. The mechanism by which administration of a small dose of endotoxin in our normal subjects produced an increase in the serum neopterin level is as yet undetermined. A recent study noted that of several inducers of mac50 ::J .....

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Figure 1. Serial determinations of serum neopterin levels in five normal subjects after the administration of intravenous endotoxin.

Neopterin levels after i.v. endotoxin . 321

rophage actlVlty in vitro, including endotoxin, only interferon-y (and interferon-a to a much lesser degree) stimulated neopterin production by macrophages (27). However, other investigations have reported the production of neopterin by macrophages in vitro when these cells were treated with endotoxin alone (28, 29). It has been suggested that in human peripheral blood monocytes the action of endotoxin is mediated by lymphocyte factors (29). Another study has noted that murine T-Iymphocytes primed by interleukin-2 in vitro will produce interferon-y when stimulated with lipopolysaccharide (30). In addition, a non-T-cell mechanism in mice for the production of interferon-y, after stimulation by lipopolysaccharide, has been described (31). Investigators evaluating cytokine production after the administration of endotoxin to normal human subjects have not found an increase in circulating interferon-y (23, 24). This may relate in part to the sensitivity of the assay procedure and to local production of interferon-y that is not reflected in systemic measurements. The delayed rise of neopterin levels described in our subjects post-endotoxin administration supports the proposal that the production of neopterin in vivo is not a direct effect of endotoxin and more likely results from the endogenous mediators released in response to the endotoxin (29). Neopterin levels have been correlated with survival in patients with sepsis (17). The neopterin levels of our subjects were comparable to levels measured in survivors of septic shock (17). Endotoxin administration to normal subjects qualitatively reproduces the hemodynamic profile of septic shock and provides a model of the earliest responses that occur during severe infections (22). In contrast to a single dose of endotoxin, serious infections result in higher and more sustained exposure to not only endotoxin, but other bacterial products which result in intense host inflammatory responses culminating in shock. Following the inflammatory events initiated by a single dose of endotoxin, normal humans develop a delayed, but sustained, rise in neopterin levels which persist well after the acutephase response has subsided. References

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