Anti-interleukin-l strategies in the treatment of the septic shock syndrome

The septic shock syndrome has recently been termed the systemic inflammatory response syndrome (SIRS). It is an acute illness characterized by generalized activation of the endothelium. The most severe and most common form of the syndrome is found in patients wilh shock due to Gram-negative sepsis. In this overview. both animal and limited human data are considered for the contribution of the cytokine interleukin-1 (IL-1) to this syndrome. Cytokines are small molecular weight, endogenously produced proteins with multiple biological effects. Laboratory investigations suggest that IL-1 plays a critical role in SIRS by promoting the biochemical and clinical characteristics of SIRS. The biochemical changes induced by IL-1 are similar to those of tumour necrosis factor (TNF) and include increased synthesis of nitric oxide, prostaglandins, platelet activating factor and endothelial cell adhesion molecules. Togelher, IL-1 and TNF appear to act in a synergistic fashion in a variety of disease models. particularly SRIS. Specific blockade of IL-1 using soluble IL-1 receptors or IL-1 receptor antagonist suggests that blocking this cytokine may be useful in treating human SIRS.

P ATIENTS WIT!I GRAM-NEGATIVE SEPSIS AND 'SEPTIC SHOCK' continue to be at risk for death despite antibiotics and supportive care.The constellation of hypotension, coagulopathies and organ failure characteristic of •septic shock' is also present in some patients without iniection .A common pathophysiological mechanism(s) which explains the systemic inflammation of these patients is generalized endothelial cell activation.As defined by Bone (1), the term •systemic inflammatory response syndrome• (SIRS) provides a more inclusive nomenclature.The most severe form of SIRS is the shock syndrome associated with Gram-negative bacteremia, but patients with less severe (noninfectious) forms of the syndrome may benefit from the same therapies.These Lherapies now include blocking cytokines.Certain cytokines stimulate endothelial cells from their normal anticoagulant.state to a procoagulant state wilh increased adhesiveness for leukocytes and platelets.
There are now over 30 different.cytokines, and the names assigned to each reflect a prominent.biological property.In some cases where lhere are multiple biological properties, the term 'interleukin' (IL) is used.followed by a number assigned chronologically.Many cytokines are growth factors such as fibroblast and endothelial cell growth factors.some are hemopoietic growth factors, whereas others possess antiviral activity and are called interferons.Most cytokines are produced primarily in the presence of disease or immunization and contribute to in1mune responses, inflammation and endothelial cell activation; others are involved in tissue repair.
There are many steps leading to lhe development of SIRS.As discussed above.generalized endothelial cell activation is a common pathological process in SIRS.Two cytokines play a major role in the pathogenesis of the syndrome: tumour necrosis factor (TNF) and IL-1.There is also a role for interferon-yin that this cytokine increases the production of and sensitivity to TNF and IL-1 (2).All lhree cytokines activate endothelial cells.Tissue inflammation associated with SIRS likely involves another cytokine, IL-8.IL-8 is an example of the family of chemotactic peptides, which at.tracts neutrophils, monocytes and lymphocytes to pass from the circulation into tissues, stimulates lhe release of enzymes and hist.aminefrom phagocytes and mast cells, induces platelet activating factor (PAF) (3) and contributes to local tissue damage (4).
Nearly each biological properly of TNF has also been observed with IL-1.These include fever (5).sleep, hemodynamic shock (6), the induction ofprostaglandin (PG) and collagenase synthesis (7), bone and cartilage resorption, inhibition of lipoprotein lipase (8) and increased synlhesis of hepatic acute-phase proteins (9).The shock-like responses to TNF and IL-1 likely reflect their effects on lhe vascular endothelium.Figure 1 illuslrates the effects of IL-1 and TNF on endothelial cells.

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The production of TNF and IL-1 is important in SIRS, especially shock due Lo severe infection, ie, sepsis.The evidence is derived from studies showing: first.that injection of IL-1 or TNF into animals or humans causes a fall in systemic blood pressure and coagu lopathy often seen in SIRS (6,8,10-14); second, that blood levels of these cytokines are oft.enelevated in patients with the syndrome (8.11); and lhird, that specific blockade of the act.ion of these cytokines in animals wilh a shock-like syndrome reduces mortality (8 , 15-1 7).
Numerous studies imply a role for TNF and IL-1 in the palhogenesis of disease.However, a distinction should be made between the local action of these cytokines and lheir systemic effects.For example, whereas TNF and IL-1 cause hypotension, IL-8 does not.IL-8 atlracts and activates neulrophils, which are responsible for tissue inju1y.The ultimate function oft.he host defence system is the elimination of the invading organism , foreign antigen or neoplastic cell , whether by phagocytosis and antibody formation as is the case in most iniections, or lhe induction of cytotoxic T cells for elimination of malignant and virally infected cells.
Inflammation is a consequence for an efficient host defence system.TNF, IL-1 and the IL-8 family are often grouped together and called 'proinOammatory cytokines' to distinguish them from 'anti-inflammatory cytokines'.These latter cytokines are IL-4, IL-10 and Lransformlng growth factor-~.They are considered 'antiinflammatory' because when administered to animals with infection or inflammation, they reduce the severity of disease and reduce the production of IL-1 and TNF (18).
When injected into experimental animals, either TNF or IL-1 induces hypot.ensionand shock; however, when injected together, this combination acts synergistically (6).Synergism between IL-1 and TNF has been documented in several models of disease, for example, destruction of the insulin-producing beta cells in the islets of Langerhans ( 19).A single int.ravenous injection into patients with cancer of TNF (10) or IL-1 (14.20) induces a sudden fall in blood pressure often requiring treatment.Healthy volunteers receiving TNF also respond Lo it in a similar fashion (12,21).

PRODUCTION OF TNF AND IL-1 IN DISEASE
Several studies have shown that in patients with SIRS, there is a positive correlation bet.ween increasing circulating TNF and IL-1 levels and the severity of disease.Interferon-a or -y does not correlate with disease severity (22).However, systemic levels ofTNF or IL-1 are not always elevated in these patients compared with olher cytokines (2), for example, the cytokine IL-6.Plasma IL-6 levels correlate positively with disease severity, particularly in sepsis (23).despite the fact that this cytokine does not produce a shock-like syndrome in animals or humans (24).
In patients with acute infection, particularly bactere- mia, circulating IL-1 and TNF can sometimes be below the detection limits of the assays.Why are circulating levels of these cytokines not high even in the presence of severe disease?The most likely reason is that TNF and IL-1 are only transiently elevated.For example, in humans given an intravenous injection of endotoxin (25,26).levels of TNF are elevated for 60 to 120 mins despite a 6 h duration of fever .lL-1 is minimally elevated for only 60 mins.Therefore, a single blood sampling may miss the elevation of either cytokine in the circulation.In severe SIRS in some individuals, the levels of IL-1 fall as the disease worsens (27).Bioassays for these cylokines can be misleading since naturally occurring inhibitors of each cytokine interfere with activity.For TNF these are soluble TNF receptors (28): for IL-1 these can be inhibitors (29).receptor antagonists (30) or soluble receptors (31) .TNF can be bound to its soluble receptors and not be detected by eiU1er bio-or immunoreactive assays (32).In general.the role for either TNF or IL-1 as mediators of disease is not derived from studies on circulating levels in disease , but rather from specific blockade of either cytokine in animal models of disease.
The local effects of TNF and IL-1 appear to mediate neutrophil emigration.lower pain threshold and release secondaiy lipid-derived mediators such as PAF, PGs and leukotrienes.The IL-8 family of neutrophil and monocyle chemolact.icpeptides is important in inflamma-tion, and IL-1 and TNF are potent inducers of IL-8 synthesis from monocytes, fibroblasts and endothelial cells.Concentrations of TNF or IL-1 as low as 1 lo 10 pg/mL will induce IL-8 production from cultured cells (33).Therefore , because of the potency of TNF and lL-1.local inflan1lllalion is likely mediated , in part.through the induction of IL-8.IL-8 stimulates the release of enzymes from neutrophils and these enzymes, in turn.break down tissues (4).In addition.IL-8 activates neutrophils to generate reactive oxygen radicals , leading lo tissue damage.The endothelium be om.es'leaky' after exposure lo IL-8 -aclivated neutrophils.

TNF AND IL-1 AS MEDIATORS IN INFECTION
A major teaching is lhal microorganisms bring abou l hypotension in sepsis by their direct effect on the vasculature.Most bacterial invasion is associated with the production of toxins that, upon entrance into the circulation, cause hypotension.decrease perfusion of vital orgai1s, acidosis and death.These toxins can be lipopolysaccharide endoloxins from Gram-negative bacteria and a large number of various exotoxins from Gra111 -positive and Gram-negative bacteria.
As recently reviewed (2).endotoxins and exoloxins can directly contribute lo hypotension by activating complement.Cell wall materials other than lipopolysaccharide from bacteria and fungi can also activate complemen l.Activated complement induces the release of PAF, PGs and nilric oxide, each a potent vasodilator.In addition , microorganisms and their toxins can indirectly cause hypotension by inducing TNF and IL-1 which , in tum , induce PAF (34,35).PG (36) and nitric oxide (37).

Strategies for reducing the effects of IL-1 in disease
The best example of toxin-mediated is •toxic shock syndrome' associated with special strains of Staphylococcus aureus.This organism produces a potent exotoxin.However, this 'toxin' is not., in the traditional sense, damaging to cells, but rather exhibits 't.oxicity' due to its ability to induce TNF and IL-1 (38).Injections of the toxin into animals results in a shocklike st.ate with elevated levels of TNF and IL-1.Patients dying with the staphylococcus toxic shock syndrome rarely have evidence of bacteremia.Streptococcal toxic shock syndrome is also due to an exotoxin that induces TNF and IL-1 production (39).
The first evidence that bacteremias or toxemias (either Gram-negative or Gran1-positive) were lethal via the production of cytokines came from a study showing that neutralizing antibodies to TNF prevented death in mice following a lethal injection of endotoxin (40).That experiment clearly established that specifically blocking a cytokine could prevent a host-mediated selfdest.rucUveprocess.The conclusion from that study was that infectious organisms (or !.heir toxins) induce the host.t.o make a lethal amount of TNF.Subsequent studies showed that.blocking TNF wit.h monoclonal antibodies reduced deaths in baboons given a lethal inject.ion of Escherichia coli organisms (41).or prevented endotoxin-induced hypotension in rabbits (42).Similar dat.a show that.blocking IL-1 also prevents lethal shock in mice, rabbits or baboons (43)(44)(45)(46).
TNF-cx has been injected into human subjects.and many of its systemic effects, such as fever , leukopenia and hypotension .which were first reported in animals, have also been observed in humans (10, 12,21.47,48).When human volunteers are injected int.ravenously with endotoxin.t.he rapid fall in blood leukocyte counts correlates directly with the peak level of plasma TNF (25).TNF induces an immediate fall in circulating neu-12A trophils in healthy humans, followed by a leukocytosis (21).A single intravenous injection of TNF results in activation of several clot.Ung parameters, including an increase in plasminogen activator inhibitor levels (12).These and other aspects of the human response to TNF are similar to !.hose observed in acule infect.ionand systemic inflammation.
IL-1 has been administered t.o human patients wit.h melanoma in phase 1 trials.Intravenous adminislrat.ion of IL-1 from 10 to 300 ng/kg has produced fever, anorexia, generalized myalgias, arthralgias, headache and hypolension (14,20,49).Moderate lo severe hypotension has been reported with doses over 30 ng/kg.The subcutaneous roule is associated with fewer side effects.These clinical studies confirm animal experiments showing !.he neulrophil-inducing property of IL-1.An increase in circulating platelets has also been observed in humans following IL-1 adminislraUon (49) .

Inhibition of synthesis or processing of IL-1 :
The slrat.egies being investigated in att.empt.slo reduce !.he production or acUon of IL-1 are shown in Table 1.A number of drugs, cytokines and other substances inhibit IL-1 and TNF production.but in a nonspecific fashion since the production of at.her cytokines is inhibited as well.Inhibit.ion of the cleavage step required lo generate an active form of IL-1 is a pot.enlial mechanism for reducing IL-1 activity (50,51).Specific receptor blockade using IL-1 receptor antagonist: Naturally occurring substances !.hat specifically inhibit.IL-1 have been detected in various human body fluids.Of these substances.the most.studied and well characterized has been !.he '1Ll inhibit.or'described by Jean-Michel Dayer and William Arend.The IL-1 inhibitor compel.eswith the binding of IL-1 t.o its cell surface receptors (30).Because of this mechanism of act.ion.'1L-l inhibitor' was renamed IL-1 receptor antagonist (IL-lRa).
Administration of IL-lRa to animals significantly reduces the severity of diseases, including those associated with infections.This topic has recenlly been reviewed (15,52).In mice, rabbits or baboons injected with lethal doses of endot.oxinor E coli, prior administration ofIL-lRa reduces death (13,(43)(44)(45).IL-lRaalso blocks the shock-like state due t.o Staphylococcus epidermidis (53).In many of these disease models, local inflan1mation mediated by IL-8, particularly in the lung.plays a key role.The ability of IL-lRa to b lock endotoxin-induced IL-8 production (33) may be a major component of the anti-inflammatory property of IL-lRa.Administration of IL-lRa to humans: In a phase 2 trial, patients with SIRS being treated with antibiotics and supportive care received either a placebo infusion or an infusion of1L-lRa (133 mg/h) for 72 h.The 28-day mortality was 44% in the placebo group and 16% in !.he IL-lRa group (P<0.015)(54)

pied, thus preventing bona fide IL-1 from binding to the same sites. Soluble IL-1 receptors (IL-1 R) (extracellular portions of IL-1 receptors) bind IL-1 and prevent the cytolcine from interacting with cell-bound receptors. Soluble IL-1 receptors act as antibodies to IL-1
phase 3 trial of1L-lRa in 901 patients with SIRS has been completed.The overall mortality in patients receiving placebo was 34%, and 29% in the patients receiving a high dose IL-lRa (2 mg/kg/h for 72 h) .However, in severely ill patients entering the study with a predicted mortality of 24% or greater, the 28-day mortality was 44.6% in the placebo group and 35% in patients receiving high dose IL-lRa (P=0.032 by Wilcoxon) (55).These latter data sim ilar to those reported in the phase 2 trial, and suggest further thal b locking IL-1 with high dose IL-lRa is effective in reducing deaths due to SIRS as the severity of the disease worsens.Assuming that this and other trials of IL-1 Ra show improvement in this paUenl population, blocking IL-1 may become an accepted therapeutic modality in some patients with SIRS and other severe acute diseases.
Animal experiments demonstrate that despite an excess of 100 molecules of IL-lRa lo one molecule of 11,-l, increasing this ratio from 1000 or even 10,000 of IL-l Ra lo one of IL-1 resulls in in1proved survival rates (43,44).In animal (43,45,46) and human (56) studies, the concentration of IL-lRa that is effective is 20 to 25 µg/ mL.These high levels of IL-1 Ra are needed to block nearly all IL-1 receptors.In vilro studies show that occupancy of as few as 5% of IL-1 receptors by IL-1 is su fficient lo trigger a b iological response (18) .Therefore, increasing the plasma concentration of IL-I Ra to 20 µg/mL insures nearly complete b lockade of endothelial cell IL-1 receptors.Balance of IL-1 and IL-lRa production: During experimental endotoxemia in humans or in patients with SIRS, levels of IL-1 Ra are 100-fold greater than levels of IL-1 (57 ,58) .It is unclear whether these endogenous levels of IL-lRa are effectively blocking IL-1 since blood levels in humans being treated with IL-1 Ra are 20 µg/mL, whereas the concentrations of IL-lRa thal circulate in human disease slates are rarely over 1.0 lo 2 .0ng/mL -a 10,000-fold difference.Nevertheless , the balance between the agonisl, IL-1.and the antagonist, IL-lRa, may determine outcome in some diseases.For example, leukemic cells from patients with acute myelogenous leukemia do not express the gene for IL-lRa, whereas there is spontaneous expression ofIL-1 in these same cells (59).High levels of IL-1 Ra in the joint fluids of patients with Lyme arthritis correlate with a shorter duration of inflammation compared with pat.ienls with low levels of the antagonist (60) .

IS THERE AN ADVANTAGE TO BLOCKING IL-1 AND TNF AT THE SAME TIME?
Although IL-1 and TNF' act in a synergistic fashion.blocking either TNF or IL-1 reduces the severity of acute disease in a variety of animals.Limited in vitro studies suggest that blocking IL-1 and TNF' al the same time is even more effective than blocking each separately: however, this dual blockade has not been studied in animal models and thus it remains speculative whether blocking both cytokines al the same time will improve outcome over those of blocking IL-1 or TNF' separately.There are no clinical studies al present to examine the effectiveness of combined therapy.
In lhe case of SIRS.blocking IL-1 or TNF' may not be beneficial in some circumstances.First, once these cytokines are produced and once they trigger their respective receptors.il will be Loo late Lo reduce their effects.For example, after IL-1 or TNF' stimulate PAF'.PG or nitric oxide, blocking the cytokines will not reduce the expression of these critical small mediator molecules.Second, in the case of activation, lhe induction of PAF', PG and nitric oxide could be direct and bypass a requirement for IL-1 and TNF' mediation.Finally, il is possible that both IL-1 and TNF' need Lo be blocked at the same Lime for a benefit to be observed.

DOES BLOCKING IL-1 ENDANGER HOST IMMUNE
AND NATURAL DEFENCE SYSTEMS? ls il possible that blocking lhese cylokines in some pa lien ls worsens their disease?Administering IL-1 Ra or anli-TNF antibodies to healthy animals has nol revealed evidence of decreased immunological function.In a phase 1 clinical trial, healthy volunteers received a infusion of IL-lRa for 3 h, and there was no evidence of impaired host defence mechanisms (56).Similar findings have been reported for monoclonal anti-TNF' (63).Thus, short term blockade ofTNF' or IL-1 appears to be safe.
The remaining question is whether a more prolonged blockade will interfere with fundamental host defence mechanisms, particularly during disease slates.Antibodies Lo TNF' have a longer half-life in the circulation than TNF' soluble receptors have and, therefore, may have greater effects on host defence.There is a large body of evidence showing that eith r IL-1 or TNF', particularly at low concentrations, increases natural resistance in animals (64,65).In some animal models.administration of antibodi s to TNF has not been effective in reducing severity of dis as (66).and blocking TNF' suppresses immune functions (67).Similarly, blocking IL-1 in some animal models increases mortality (68,69).Therefore, contrary to most animal data discussed above, some models reveal a worsening of disease when blocking TNF' or IL-1.If blocking IL-1 or TNF' in disease impairs host defence systems, then clinical trials in patients with SIRS may show an increase in mortality associated with this U1erapeutic approach.IL has been reported in the lay pr ss that in large scale studies of patients with SIRS, a subgroup of patients (those without bacleremia) apparently have increased mortality when treated with either anticore endoloxin (HA-lA) or monoclonal anli-TNF' antibodies, compar d with patients treated with placebo.AL present, there are no rapid, predictive tests Lo determine which patient will benefit and which patients might worsen during cylokine blockade.

Figure 2 )
Figure 2 ) Cell-bound receptors for inte rleulcin (IL) -1 are bloclced by monoclonal antibodies (MoAb).which prevent binding of IL-1 lo the extracellular portions of the receptors.No signal istransduced to the nucleus .In the presence of excess IL-1 receptor antagonist (IL-1 Ra) IL-1 binding sites on the receptors are occu- CAN J INFECT DIS V OL 5 SUPPL A FEBRUARY 1994 Y•DO OTCCl'Y Blocking IL-1 in septic shock

TABLE 2 Reduction in severity of animal disease activity Disease model
(62)ble receptor is another lerm to describe the extracellular portion of a receptor.The extracellular portion of the IL-1 RI binds and neu -Lralizes IL-1 in a fashion similar lo antibodies to IL-1.When soluble IL-1 type I receptors were given to mice undergoing allograft hearl transplanlalion, survival of mice was increased(62).The effects of the soluble type I receptor are likely due to decreased inflammation rather than decreased immunoresponsiveness. Soluble human IL-lRI is in phase 2 clinical trials for SIRS.Figure2illustrates the various mechanisms for blocking IL-1.
Table 2 lists the effects of IL-1 antagonism in animal models.