Hypoxanthine derivatives in experimental infections

C DE SIMoNE, EA MARTELU, P FoRESTA. et al. Hypoxanthine derivatives in experimental infections. Can J Infect Dis 1992;3(Suppl B): 106B-110B. In vivo treatment with parenterally administered hypoxanthine derivatives. notably ST 789, was able to protect cyclophosphamide-immunosuppressed mice against experimental infections with both bacterial and fungal pathogens. However, the mechanisms accou nting for these effects of hypoxanthine derivatives remain to be fully established. In fact. only the treatment with ST 789 resulted in a clear enhancement of the primary antibody production as well as macrophage phagocytic activity, whereas T lymphocyte responsiveness to mitogens and both macrophageand natural killer-dependent cytotoxicity were not significanUy affected. These data. together with the recently shown ability of ST 789 to increase interleukin-6 production, suggest that monocyte/macrophages are likely to be the main cellular target of the imrnunomodulaUng activity of ST 789. Finally, in the presentln vivo study, hypoxanthine derivatives did not enhance the mean survival lime of tumour-bearing immunosuppressed mice.

Several arginine-containing hypoxanthine derivatives are currently available.The main difference between ST 789 and its derivatives is that the derivatives, instead of having a terminal L-arg aminoacidic residue, possess an L-arg terminal peptide of varying length (two to five aminoacidic residues).However, reports evaluating both in vitro and in vivo immunomodulating properties of the derivatives are currently lacking.In contrast, ST 789 has been evaluated extensively in this respect.The present study found that, as a group, hypoxanthine derivatives other than ST 789 are not very potent immune modulators.However, after treatment with many hypoxanthine derivatives (including ST 789) experimentally infected mice were protected against challenge with several pathogens.Finally, while ST 789 had some antitumour activity, the other derivatives evaluated did not
The protective effects of combined treatment with ST 789 and ceftazidime.and ST 789 and amphotericin B were also evaluated in cyclophosphamide-treated mice challenged with K oxytoca and C albicans, respectively.Ceftazidime and amphotericin B were administered at per se completely ineffective dosages ranging from 0.02 to 2.0 mg/kg and 1.0 to 2.5 mg/kg, respectively.
The statistical analysis of results was performed using Fisher's test and x 2 test.
Primary antibody production: B6D2F1 mice (age six weeks) were administered hypoxanthine derivatives intraperitoneally at either 0.25, 2.5 or 25 mg/kg per day for five days.On day 3, mice were injected with 1xl0 7  sheep erythrocytes in 0.2 mL.Spleen cells obtained from mice immediately following death were adjusted at a density of 1xl0 7 cells/mL in Iscoves' modified Dulbecco's medium (IMDM; Gibco, New York) supplemented with fetal calf serum, 5x10-5 M 2-mercaptoethanol, 4% polyethylene glycol (MEG; Serva, Germany) (molecular weight 6000 Da) and 50 U /rnL of both penicillin and streptomycin.Antigen was added in the form of 2 rnL 10% sheep red blood cells/mL of culture.Control cultures without antigen were set up in several experiments.The cultures were incubated at 37°C in 5% carbon dioxide for 90 mins.The cells were washed, resuspended in medium and assayed for plaque-forming cells by using the previously described slide modification of the hemolysis in gel method (14).
T lymphocyte proliferation to mitogens: T lymphocyte proliferative responsiveness to mitogens was assayed as previously described (15).Spleen cells were obtained from mice given five days of therapy with hypoxanthine derivatives.Delayed T hypersensitivity reactions: Delayed T hypersensitivity reactions were assayed as previously reported (16).Macrophage-dependent cytotoxicty: Peritoneal exudate cells were obtained by washing the abdominal cavity of B6D2F1 mice with RPM! 1640.Harvested cells, were centrifuged at 400 g for 10 mins at 4°C and density adjusted to 2x10 6 cells/rnL.Daudi cells were used as target cells.The cytotoxicity test was performed as previously described (5).Phagocytic activity: The phagocytic activity of peritoneal exudate cells was assayed as previously reported (17).Briefly, peritoneal exudate cells were adjusted to a density of 4X10 6 cells/mL to which was added sheep red bloods cells previously opsonized with anti-sheep erythrocyte immunoglobulins.After 1 h incubation at 37°C in 5% carbon dioxide, nonphagocytosed erythrocytes were removed using hypotonic saline solution.
Natural killer cell-dependent cytotoxicity: The assay of natural killer cell activity was performed using standard methods (18).Antitumoral activity: Antitumoral activity was assayed as previously described (19).Br;efly, inbred C57B1/6 (age eight weeks) and DBA/2 (age six weeks) male mice were used in the experiments with both carcinoma and leukemia models.Outbred CD 1 male

Experiment al infections:
All hypoxanthine derivatives tested were able to protect cyclophosphamide-treated mice against experimental infections with both bacterial and fungal pathogens as shown by improvement in both percentage mortality and mean survival time.In the K oxytoca, S marcescens and S typhimurium infective models, ST 789 was most effective when administered intraperitoneally.as it proved active at concentrations as low as 0.25 mg/kg (Tables 1,2).Similar results were obtained in K pneumoniae, E coli and Ps mirabilis models (data not shown).
In contrast.subcutaneous administrations of the immunomodulator were able to protect mice against only P aeruginosa and C albicans infections, the former occurring when ST 789 was administered alone or in the association with gentamicin (data not shown).and the latter occurring when ST 789 was combined with an 'ineffective' dosage of amphotericin B (

DISCUSSION
The results clearly show that hypoxanthine derivatives, most notably ST 789, exhibit immunomodulaling properties.In fact, this group of molecules protected cyclophosphamide-immunosuppressed mice against experimental infections with bacterial and fungal pathogens, confirming previous reports (10,11).Furthermore, the combined administration of ST 789 and antimicrobial agents, such as ceftazidime, amphotericin B and gentamicin.at doses per se completely ineffective, strongly protected cyclosphosphamidetreated mice in several models of experin1ental infections.Therefore, ST 789 either alone or in combination with antibiotics enhances host resistance to pathogens.
The mechanisms responsible for these effects of hypoxanthine derivatives are unclear.The reported ability of ST 789 to restore mitogen responsiveness of lymphocytes from cyclophosphamide-treated mice is likely to play a major role (21).However, other cells could mediate the immunomodulating activity of ST 789.In fact, ST 789 treatment expanded bone marrow natural killer progenitor cells in vivo, thus strongly enhancing the regeneration of mouse natural killer cell activity (9).Furthermore, as cyclophosphan1ide treatment severely reduces the number of circulating granulocytes (22), the capacity of hypoxanthine derivatives to protect cyclophosphamide-administered mice against pathogens might -at least in part-depend on their myelorestorative ability.However, a role for colony stimulating factors in mediating the immunomodulation of hypoxanthine derivatives is still unproven.
Data from the authors' laboratory showing an increased in vitro production of IL-6 by peripheral blood mononuclear cells cultured in the presence of both mitogens and ST 789 compared with control cultures suggest that hypoxanthine derivatives could act in vivo by modulating cytokine production (6).Furthermore, the present report shows that in vivo administered ST 789, as well as other hypoxanthine derivatives, strongly enhanced the phagocytic activity of peritoneal exudate macrophages.Since macrophages are the main cellular source of IL-6 (23) this suggests that hypoxanthine derivatives exert an action on monocytes/macrophages.Overall, these findings suggest that hypoxanthine derivatives could modulate infections with intracellular pathogens such as listeria, legionella, brucella, salmonella and mycobacteria.
A temporal relationship in cytokine production has been recently demonstrated for tumour necrosis factor, IL-l and IL-6 production (24).After a bacterial challenge, the appearance of IL-6 follows that of tumour necrosis factor and IL-2 during endotoxemia (25,26).Thus, the most likely scheme of activation leading to cytokine production involves the early release of tumour necrosis factor and IL-l from monocytes/macrophages which subsequently induces the secretion of IL-6 and other cytokines from both monocytes and structural cells, such as fibroblasts and endothelial cells.This cascade is the basis for the finding of sequential elevations of cytokines in plasma in experimental models of inflammation (25)(26)(27).
The role of tumour necrosis factor in mediating the ST 789-dependent increase of IL-6 production remains to be established.Furthermore, there is no report on the ability of hypoxanthine derivatives other than ST 789 to modulate cytokine release in vivo or in vitro.In this study, these compounds exhibited immunomodulating activity in vivo.However, the in vitro lymphocyte responsiveness to mitogens, natural killer cell activity and delayed T cell hyperreactivity reactions were not significantly affected.Only ST 789 was able to increase the primary in vitro antibody production compared with other hypoxanthine derivatives.possibly by increasing IL-6 production (6).Therefore, the mechanisms involved in determining enhanced host resistance by hypoxanthine derivatives are sWl unresolved.
Finally, in the present study hypoxanthine derivatives did not show any in vivo antitumour activity.The results are in contradiction with previous reports in-dicating that mice injected with ST 789 exhibited significant resistance against two synergistic tumours (7).Furthermore, the arginine residue of ST 789 has been suggested to play a pivotal role in mediating the antitumour activity of ST 789 by increasing T lymphocyte cytotoxicity (3,4) as well as natural killer cell and macrophage cytotoxicity (3)(4)(5).The enhancement ofT helper function could also be involved, as suggested by the absolute increase of CD4+ T cells observed in cancer patients following treatment with arginine-lysine combination (unpublished data).The inability to find any increase in mean survival time of tumour-bearing mice following in vivo administration of hypoxanthine derivatives may be related to peculiarities of the experimental protocol.
CAN j INFECT DIS VOL 3 SUPPL 8 AUGUST 1992 0 LY • DO OT CQrlxanthine derivatives and infections CAN J INFECT DIS VOL 3 SUPPL B AUGUST 1992 0 • oxanthine derivatives and infections

Table 3 )
. Oral ST 789 administration was completely ineffective.Dosages per se completely ineffective of ST 789 and ceftazidime significantly protected, in terms of survival, immunosuppressed mice from experimental infection 1088

TABLE 3 Protective effects of combined treatment with ST 789 and amphotericin B in cyclophosphamide-immunosup- pressed mice challenged with Candida albicans
ST 789 administered subcutaneously daily, five days prior to infection.Amphotericin 8 administered by single intraperitoneal injection two days after infection.'Untreated, cyclophosphamide-immunosuppressed (750 mg/kg) five days before infection) mice infected with 5x1d' C albicons cefls."P<O.OD/ (Fisher's test)

TABLE 4 Protective effects of combined treatment with ST 789 and ceftazidime in cyclophosphamide-immunosup- pressed mice challenged with Klebsiella oxytoca 552 Number of deaths/ ST 789 Cettazidime total number of (mg/ kg/ day
with K oxyLoca (Table4).The same was true in the A fumigaLus model since ST 789 and amphotericin B administered at dosages per se ineffective were synergistic (data not shown).