Role of Oxygen in PhagocyteMicrobicidal ActionRobert C. AllenResearch and Development Division, ExOxEmis, Inc., Little Rock, Arkansas

Immune information in the form of inflammatory mediators directs phagocyte locomotion and increases expression of opsonin receptors such thatcontact with an opsonized microbe results in receptor ligation and activation of microbicidal metabolism. Carbohydrate dehydrogenation and 02 con-sumption feed reactions that effectively lower the spin quantum number (S) of 02 from 1 to 1/2 and finally to 0. Oxidase-catalyzed univalent reduc-tion of 02 (S = 1; triplet multiplicity) yields hydrodioxylic acid (HO2) and its conjugate base superoxide, 0° (S = 1/2; doublet multiplicity). Acid orenzymatic disproportionation of superoxide yields H202 (S = 0; singlet multiplicity). Haloperoxidase catalyzes H202-dependent oxidation of Cl- yield-ing HOCI (S = 0), and reaction of HOCI with H202 yields singlet molecular oxygen, 102 (S = 0; singlet multiplicity). The Wigner spin conservation rulerestricts direct reaction of S = 1 02 with S = 0 organic molecules. Lowering the S of 02 overcomes this spin restriction and allows microbicidal com-bustion. High exergonicity dioxygenation reactions yield electronically excited carbonyl products that relax by photon emission, i.e., phagocyte lumi-nescence. Addition of high quantum yield substrates susceptible to spin allowed dioxygenation, i.e., chemiluminigenic substrates, greatly increasesdetection sensitivity and defines the nature of the oxygenating agent. Measurement of luminescence allows high sensitivity, real-time, and sub-strate-specific differential analysis of phagocyte dioxygenating activities. Under assay conditions where immune mediator and opsonin exposure arecontrolled, luminescence analysis of the initial phase of opsonin-stimulated oxygenation activity allows functional assessment of the opsonin recep-tor expression per circulating phagocyte and can be used to gauge the in vivo state of immune activation. - Environ Health Perspect 1 02(Suppl 10):201-208 (1994)

Key words: phagocyte, spin conservation, microbicidal, combustion, oxygenation, luminescence, lucigenin, luminol, haloperoxidase, opsonin,opsonin receptor

Phagocyte Reduction andOxygenation ActivitiesPhagocyte microbicidal action is adynamic, metabolically linked processwhereby reduction potential provides thedriving force to convert molecular oxygen(02) into effective oxygenating agents.Increased phagocyte metabolism of glucosethrough the dehydrogenases of the hexosemonophosphate shunt in combinationwith increased nonmitochrondrial 02 con-sumption is collectively referred to asphagocyte respiratory burst metabolism(1-3). Glucose dehydrogenation generatesthe equivalents for univalent reduction of02' that in turn initiate the transducingreactions required for phagocyte generationof oxygenating agents. Microbe killingresults from the reactions of these metabol-ically generated oxygenating agents withmolecular components of the target microbe.

On initial consideration, the mobiliza-tion of reduction potential might seem anunlikely initial step in the direction ofphagocyte generation of oxygenatingagents. This apparent paradox is resolvedby taking a quantum mechanical perspec-

This paper was presented at the Conference onOxygen Radicals and Lung Injury held 30 August-2September 1993 in Morgantown, West Virginia.

Address correspondence to Robert C. Allen, 3215Woodcrest, San Antonio, TX 78209-3138. Telephone(210) 822-5091. Fax (210) 822-0948.

tive. As predicted by the Pauli exclusionprinciple, the 02 we breathe is a diradicalmolecule with one electron occupying eachof its two pi antibonding (7*) orbitals, andaccording to Hund's maximum multiplic-ity rule, both electrons have the same spinquantum number (s), i.e., both electronswill have an s value equal to 1/2 or -1/2(4). As such, the total or net spin quantumnumber (S) of 02 is either 1 or -1.Multiplicity is a spectroscopy expressionrelated to S by the equation 12S1+1. Thus,in its lowest energy or ground state, 02 is atriplet multiplicity diradical molecule, i.e.,12(1)1+1=3, and is highly paramagnetic (5).

Spin ConservationSymmetry must be conserved in all chemi-cal reactions. The Wigner spin conserva-tion rules define the tendency of a reactingsystem to conserve spin angular momen-tum; i.e., the total orbital angular momen-tum of all the electrons of a reacting systemis conserved (5-7). This conservation prin-ciple is illustrated by the Wigner-Witmercorrelation rules presented in Table 1 (8).The more familiar Woodward-Hoffmanrules define the conservation of orbitalangular momentum of each electron sepa-rately. Reaction allowedness and feasibilityrequire conservation of both total and indi-vidual symmetries (9).

Reactions of organic and biologic mole-cules with 02 are typically of very high

exergonicity, but such reactions do notoccur spontaneously. Consider the heat ofreaction calculations for the reaction of 02with the double bond of ethylene (10):{C2H4 (+ 146 kcal/mole) - 2 CH2} [1]

{02 (+ 119 kcal/mole) - 2 0} [2]

{2 CH2 + 2 -*2 CH2O + (-358 kcal/mole)} [3]

Net AG = -93 kcal/mole(equivalent to an einstein of308 nm photons)

This oxygenation reaction yields freeenergy magnitudinally greater than thattypically considered in bioenergetics. Theconcept of spin conservation is at the cruxof understanding why such high exer-gonicity reactions do not spontaneouslyoccur. Ethylene, like other typical organicand biologic molecules, is a singlet multi-plicity S=O molecule, and as described inTable 1, the direct reaction of an S= 0molecule with S= 1 02 would proceed viaa S= 1 reaction surface to yield an S= 1oxygenation product. Absolute reactionrate theory predicts a low probability forany reaction involving change in multi-plicity (11,12). The transmissioncoefficient of the absolute reaction rate

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201

Table 1. The Wigner-Witmer spin correlation rules.

Reactant A Reactant B Potential energy surface

Singlet (S = 0) Singlet (S = 0) Singlet (S = 0)Doublet (S = 1/2) Doublet (S = -1/2) Singlet (S = 1/2.1/2 = 0)Triplet (S = 1) Triplet (S = -1) Singlet (S = 1-1 =0)Singlet (S = 0) Doublet (S= /2) Doublet (S = 1/2)Triplet (S = 1) Doublet S=-1/2) Doublet (S= 1-1/2=1/2)Singlet (S = 0) Triplet (S = 1) Triplet (S= 1)Doublet (S = -1/2) Quartet (S = 11/2) Triplet (S = -1/2 + 11/2 = 1)Doublet (S = -1/2) Quintet (S = 2) Quartet (S = -1/2+ 2 = 1/2)

equation gauges the spin allowedness of a

reaction (7).

Chemical CombustionWe all have a first-hand experience withthe phenomenon of burning. Hold a litmatch to a piece of paper and the paper

will burn. In this case, the match providesthe activation energy required to spread thefire to the paper, i.e., to initiate combus-tion. Although ethylene does not sponta-neously react with 02, combustion isreadily initiated with a spark. Even highlyexergonic chemical combustion can requirerelatively large activation energies. Onceinitiated, the energy liberated by reactionmaintains propagation.

Chemical combustion can be bestunderstood as a radical (S > 0) propagationreaction. Initiation of burning requires theapplication of energy sufficient to producehomolytic cleavage of the organic molecu-lar bonds, i.e., to convert the S= 0 substratemolecule ('substrate) into S=1/2 reactants

2reactants):substrate + heat -> 2 2reactants. [5]

The superscript preceding the symbol indi-cates the multiplicity of the symbolizedreactant. The doublet multiplicity (S= 1/2)radicals produced by homolytic cleavagecan react with triplet multiplicity (S= 1)302 by radical-radical orbital overlap to

yield covalent bonding,

2reactant + 302 o 2product + AG [6]

as described in Table 1. The S= 1/2 radicalproduct of this oxygenation plus the freeenergy liberated ensure additional reactionwith S= 1 triplet multiplicity 302,

2product +302 2product + AG [7]

to produce S=112 radical products as requiredfor radical propagation, i.e., continued burn-ing, until the substrate or 302 is depleted.Reaction with another S= 1/2 radical,

2product + 2reactant -- 'product + AG [8]

i.e., radical-radical annihilation (S = 1/2-1/2 = 0), terminates the reaction andyields a singlet multiplicity product as

shown in Table 1.

Microbicidal Combustion: Chaningthe Spin Quantum Number of02Phagocytes exert a broad and lethal micro-bicidal action using the simple ingredientsof metabolically generated reductionpotential and 02' The character of respira-tory burst metabolism suggests the possibil-ity that phagocyte microbicidal actionmight involve combustionlike oxygenationreactions, but are phagocytes energeticallyand mechanistically capable of changingthe spin quantum number of 02 from S= 1

to S= 1/2, and finally to S= O? Doing so

would overcome the symmetry restrictionsto 02 reactivity and, as such, the elec-trophilic reactive potential for direct 02

reactivity would be realized. Chemicalcombustion (burning) requires radicaliza-tion of substrate, i.e., increasing the valueof S, to guarantee reaction with triplet mul-tiplicity 02. Phagocyte microbicidal com-

bustion requires deradicalization of 02,

i.e., decreasing the value of S, to guaranteeits reaction with singlet multiplicityorganic molecules (13). Note that reactionby either pathway satisfies the spin conser-

vation requirements.Activation of phagocyte respiratory

burst metabolism is linked to lowering theMichaelis constant of membrane-associatedNADPH:02 oxidoreductase for its reducedsubstrate NADPH (2). The univalentreduction of 02 catalyzed by this flavopro-tein oxidase (14-17):

2 2H (NADPH + H+) + 2 302

22 2HO2 <-+2 0 +2H+[9]

is the first step in lowering the S value or

multiplicity of 02. Hydrodioxylic acid(2HO2; perhydroxylic acid) and its conju-gate base superoxide (2O), the products ofunivalent reduction, are doublet multiplic-ity S = 1/2 molecules.

Hydrodioxylic acid has a plA of 4.8,and its generation by the activated oxidasemay be a major factor in the dynamicacidification of the relatively smallphagolysosomal space (18). When the pHof the space is 4.8, the ratio of HO2 to

°2- is unity. As the pH approaches the2~~~~~~~~~~pK), the anionic repulsion that prevents

direct disproportionation of 20- is no2

longer a barrier to reaction, and as such,the doublet-doublet annihilation reaction,

2HO +20 + H+ H2O2 + 2 [10]

approaches maximum rate, i.e., 8 x I07M-1sec-1 (19). Note that Equation 10 isthe same type of radical termination reac-

tion described by Equation 8. If the reac-

tion is a direct annihilation through an S=0surface, both H202 and 02 will be in theS= 0, singlet multiplicity state as describedin Table 1 (20). On the other hand, an

indirect stepwise disproportionation involv-ing high multiplicity catalysts, e.g., superox-

ide dismutase (21), can yield the mixedspin products IH 0 and ground state 30

2 2 2

(22).Superoxide (2O-) can also participate

in doublet-doublet annihilation reactionwith nitric oxide (2NO), another S= 1/2reactant,

2NO 20- OON0- [11]

to yield the peroxynitrite anion. As predi-cated by the spin symmetry conservationrules, neither 20 nor 2NO reacts readilywith S= 0 organic or biologic molecules,but since both have a common radicalS= 1/2 symmetric character, they readilyreact with each other. The reported rate

constant for Reaction 1 1 is 4 x 107M-'sec-' (23,24). Radical annihilation viaa S= 0 surface generates a product, i.e., per-

oxynitrite, with S= 0 symmetry in com-

mon with most organic and biologicmolecules, and as such, symmetry is not a

barrier to direct reaction of peroxynitritewith these S= 0 molecules (25).

Haloperoxidase ActivityAlthough metabolically costly, the reduc-tion of 02 to H202 is necessary to elimi-nate the S= 1 character that limits its directreactivity. H202, a S= 0 molecule, can par-

ticipate in spin allowed reactions with S= 0organic and biologic molecules. However,H202 is a weak acid with a pKa of 11.65,and in its protonated form, H202 is rela-tively unreactive. The direct reactive capac-

ity of H202 increases with alkalinity, e.g.,

the Dakin reaction. At a physiologic pH of

Environmental Health Perspectives

R. C. ALLEN

202

PHAGOCYTE OXYGENATING ACTIV TY

7, the HOT/H202 ratio is approximately1/100,000 and, as such, the actual concen-tration of HO- available as a microbicidaloxygenating agent is modest.

Granulocytic leukocytes and bloodmonocytes contain haloperoxidases, i.e.,myeloperoxidase and eosinophil peroxidase,that exert a lethal microbicidal action (26).These H202:halide oxidoreductases catalyzethe oxidation of halide (X7) to hypohalousacid (HOX) and the reduction of H202 toH20 (27,28). This oxidation-reductionreactive coupling can be described in theNernst equation:

AE = RT l( [H20][HOXflnF [X- I[H202] [H]) [12]

AE is the change in potential (volts), R isthe gas constant, T is the absolute tempera-ture, n is the number of electrons pergram-equivalent transferred, F is a faraday,and In is the natural log of the reactantsand products as shown (29). The influenceof pH and type of halide on the AE andAG of the reaction is shown in Table 2.The exergonicity of either chloride (CF) orbromide (Br-) oxidation, the primary reac-tion catalyzed by haloperoxidase, increasesdirectly with proton availability. In the sec-ondary reaction of Table 2, HOX, theproduct of the primary reaction, reactswith a second molecule of H202 to yieldsinglet molecular oxygen (30). Note thatfor this secondary reaction, exergonicity isdirectly related to halogen electronegativityand inversely related to proton availability.

When [H202] is relatively low, someHOX can accumulate and react with bio-logic molecules, e.g., chloramine forma-tion. Since both HOX production andconsumption are dependent on [H202],and since the reaction of HOX withH202 is essentially diffusion controlled, itis likely that most HOX will be consumedin the secondary reaction. It is also likelythat chloramines can react with H202 toyield 102.

The first-order relaxation of 'Ag 02 toground state 302 yields a 1268-nm photon(30), and measurement of this infraredemission has been taken as direct spectro-scopic proof of 102 generation by haloper-oxidase (31). A 1268-nm emission has alsobeen reported from phorbol myristateacetate (PMA) stimulated eosinophil leuko-cytes but not from neutrophil leukocytes(32). The problem of documenting '02generation in biologic systems is illustratedby the following riddle. What is definitiveevidence for 102 generation and what is

definitive evidence that the 102 generatedhas not participated in reaction chemistry?The answer to both questions is the same:1268-nm emission. Photon emission fromchemically unreacted 102 represents wastedenergy. On the other hand, the reaction of102 with appropriate substrates can yieldhigh energy endoperoxide and dioxetaneproducts that can relax by photon emissionin the visible range of the spectrum yield-ing chemiluminescence (33). Conditionsthat maximize I02 reactivity, and thuskilling efficiency, are the same conditionswhere little or no 1268-nm emission is likelyto be observed. Introducing microbes to a1268-nm light-generating system willinhibit infrared emission in proportion tothe 102 consumed in the microbicidalreaction.

The problem of 102-specific detectionhas recently been overcome by the develop-ment of an 9,10-diphenylanthracene(DBA)-based assay for the measurement of102 in complex biologic systems. UsingDBA-coated beads and phorbol 12-myris-tate 13-acetate (PMA; 1.6 pmole/ml) asstimuli, Steinbeck et al. reported the genera-tion of 14 nmole I02/1.25 x 106 neutrophils.This quantity was equal to approximately20% of 302 consumption by the activatedneutrophils (34). Using Equations 9 and10, there is the possibility that one 102 and

Table 2. Haloperoxidase generation of hypohalousacid and singlet molecular oxygen: the effect of halideelectronegativity and pH.

Primary reactionH 0 + 1X- + H-XP0O- 1HOX + 1H20

PF4567

PF4567

EH202 - ECI = AE1 AG11.5396 - 1.3760 = 0.1636 -7.531.4805 - 1.3465 = 0.1340 -6.161.4214 - 1.3170 = 0.1044 -4.801.3623 - 1.2875 = 0.0748 -3.44

H EH202 - EBr = AE1 AG11.53961.48051.42141.3623

- 1.2130- 1.1835- 1.1540- 1.1245

0.32660.29700.26740.2378

-15.02-13.66-12.30-10.94

Secondary reaction1HOX + 1H202 - H + 1X + 02 + 1H20

pH EHOCI -4 1.3760 -5 1.3465 -6 1.3170 -7 1.2875 -pH EHOBr -4 1.2130 -5 1.1835 -6 1.1540 -7 1.1245 -

EH2020.44560.38650.32740.2683EH2020.44560.38650.32740.2683

AE20.93040.96000.98961.0192AE20.76740.79700.82660.8562

AG2-20.27-21.63-22.99-24.35AG2

-12.77-14.13-15.49-16.86

one H202 are produced per two 302 con-sumed. Based on the primary and secondaryreactions of Table 2, one 02 is expected tobe generated per two H202 consumed. IfH202 production is proportional to the 302consumed, a maximum of three 102 couldbe generated for each four 30 consumed.Thus consumption of 72 nmole of 2could yield a maximum of 54 nmole 02.The actual measured yield was 14 nmole102 for an estimated efficiency of 26%. Ifthe haloperoxidase activity described inTable 2 is solely responsible for generating10 one I0 could be generated for each2' 2two H202 reacted and by extension foreach two 302 consumed. Therefore, con-sumption of 72 nmole of 302 could yield amaximum of 36 nmole 102, and thus theestimated efficiency is 40%.

Luminescence as an Energy Product ofOxygenationsGeneration of photons in the visible rangeof the spectrum requires highly exergonicreactions. For example, 650-nm (red) pho-tons and 400-nm (blue) photons have AG'sequivalents to -44 and -72 kcal/mole pho-ton, respectively. With few exceptions, bio-chemical reactions do not yield AG'ssufficient to generate such photons.Oxygenations, 02 reactions yielding 02-containing products, are such exceptions.In the previous example (Equations 1-4),dioxygenation of ethylene was shown toyield a net AG of -93 kcal/mole. Biologicdioxygenations of carbon-carbon doublebonds yielding endoperoxide, dioxetane, ordioxetanone intermediate, and ultimatelyyielding aldehyde or ketone products, cangenerate exergonicity sufficient for elec-tronic excitation and ultimately photonemission in the visible (700-340 nm pho-tons) range of the spectrum. The transfor-mation of 02 from the S= 1 to the S= 1/2and ultimately to the S= 0 state effectivelyremoves the mechanistic barrier to dioxy-genation. Thus, the electrophilic potentialfor direct oxygenation is realized.

Luminescence is an energy product ofphagocyte microbicidal activity (13). Infact, phagocyte luminescence was firstobserved during experiments to test thehypothesis that changing the S of 02 from1 to 1/2 to 0 was required for effective 02-dependent microbicidal action. Loweringthe S of 02 from 1 to 0 would involvetransductions driven by respiratory burstmetabolism. If the resulting low multiplic-ity reactants participate in microbicidaloxygenation reactions, then it is likely thata portion of the reactions proceed viaendoperoxide or dioxetane intermediates to

Volume 102, Supplement 10, December 1994 203

R. C. ALLEN

yield excited carbonyl products that canrelax by photon emission. The observationof luminescence from activated phagocytesprovides an energy-based confirmation ofthe important role of oxygenations inphagocyte microbicidal action.

Chemiluminigenic Probing:Differential Measurement ofOxygenation ActivitiesAlthough of theoretical importance, thereare several limitations to using native lumi-nescence to quantify phagocyte oxygena-tion activity. Typically, native substratesare of relatively low luminescence quantumyield, and the nature of the substrate varieswith the microbe and the test condition. Achemiluminigenic substrate (C) is anorganic molecule susceptible to dioxygena-tion with high quantum (luminescence)yield. Introducing a C to the phagocyte testsystems increases the luminescence yieldmagnitudinally (35,36). Using a C withrelatively well-established reactive charac-teristics also defines the nature of the oxy-genating agent (X) measured (37). As such,the luminescence intensity (dLldt) is func-tionally and quantitatively linked to boththe concentration of oxygenating agent([X]) generated by the phagocyte and theconcentration ofC ([C ]) available for reac-tion:

dLIdt = k[X] [C] [13]

where k is the proportionality constant.When the [C] is much greater than the[X], the reaction order approximates zerowith respect to C, i.e., C is not rate limit-ing, and the relationship simplifies to:

dLIdt = k[X] [14]

Phagocyte oxidase function can be mea-sured as the luminescence product ofdimethylbiacridinium (DBA++; lucigenin)dioxygenation (38). Under alkaline condi-tions, i.e., as the pH approaches the pKa ofH202, lucigenin reacts directly with perox-ide,

IDBA2+ + 1HO2 (102 )

2 IN-methylacridone + photon[15]

Under neutral to mildly acid condi-tions, DBA++ can be univalently reducedresulting in change from the S= 0 to theS= 1/2 state,

IDBA2+ + e- (2Q-) _> 2DBA + (02)

In its radicalized form, 2DBA' candirectly react with 20- in a doublet-dou-2blet annihilation via a S = 0 surface,

2DBA+ +202_<2 lN-methylacridone + photon

[17]One of the two N-methylacridone pro-

duced will have an excited ketone carbonylfunction that can relax to ground state byphoton emission. Note that both the one-step peroxidation reaction and the two-stepradical intermediate reaction with 20Oyield the same product. Recall that the dis-proportionation of 20 to yield 1H2O2 issecond order with respect to superoxide,i.e., [2O-2]2. At physiologic pH, the DBA++reaction is also either second order withrespect to 20- or a mixed second orderreaction, i.e., first order with respect to [e-]and first order with respect to [20s].

DBA++ does not react with HOC1 or102 to yield luminescence and, as such,DBA++ is not a substrate for the measure-ment of haloperoxidase activity. In fact,these oxidizing and dioxygenating agentswill tend to competitively inhibit oxidase-dependent DBA++ luminescence. The netreaction by any of the above consideredpathways is:

DBA2++02+2e-2 lN-methylacridone + photon

[18]

and, as such, can be considered as a reduc-tive dioxygenation (RDOX) reaction.

The net dioxygenation of a cyclichydrazide, e.g., luminol, to yield lumines-cence does not require electrons or reduc-ing equivalents. Haloperoxidase-containingphagocytes can catalyze dioxygenation ofS= 0 luminol via the sequential two equiva-lent oxidation:

'luminol + 'HOClIdiazaquinone + 'Cl + 1H20

[19]

followed by a two equivalent reductivedioxygenation:

'diazaquinone + 'H202 <

aminophthalate + N2 + photon[20]

The possibility also exists that 102 or arelated singlet multiplicity dioxygenatingagent, e.g., 0-0-Cl-, can directly dioxy-genate luminol in a nonradical, spin-allowed manner to yield electronically

excited aminophthalate. This pathway issuggested by the electrophilic reactivity of'02 with it-systems and its tendency toform dioxetanes and endoperoxides (36).Although presently unproved, the haloper-oxidase catalyzed dioxygenation of luminolmight also proceed by the reaction:

'luminol + I02aminophthalate + 'N2 + photon

[21]

The essential relatedness of the luminolluminescence reactions catalyzed by eithermechanistic pathway can be appreciated byrecalling that the reaction of HOC1 withH202, the sequential reactants of Equations19 and 20, yields 02, the reactant ofEquation 21. Note the secondary reactionof Table 2.

Haloperoxidase-deficient macrophagesalso catalyze the luminol luminescencereaction, but the specific activity perphagocyte is greatly decreased (35).Luminol dioxygenation by macrophages(39), and possibly a portion of the activityof some haloperoxidase-containing phago-cytes, may involve NO synthase-generatedreactants such as peroxynitrite ('OONO),the product of Equation 1 1; e.g.,

'luminol + 'OONO -* laminophthalate+ NO- + N2 + photon [22]

As an additional possibility, luminolluminescence may also be catalyzed by asequential radical mechanism involving20 as a reductive oxygenating agent (36).2Such reaction requires the initial radicaldehydrogenation of luminol by a radicaloxidant such as hydroxyl radical:

luminol + (univalent oxidant, e.g., 2OH)

21uminol+ + (reduced oxidant, e.g., H20)[23]

Once generated, the univalently oxi-dized luminol radical can now participatein a doublet-doublet annihilation reactionwith superoxide:

2luminol' + 20- -* laminophthalate+ N2 + photon [24]

Evidence has been presented that classic(halide-independent) peroxidases can alsocatalyze the radical dehydrogenation ofluminol, ultimately resulting in luminoldioxygenation and luminescence (40,41).By whatever mechanism, and in contrast

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PHAGOCYTE OXYGENATINGACTIVITY

with the net DBA++ reaction describedabove, the net luminol reaction responsiblefor luminescence is a simple dioxygenation(DOX) reaction,luminol + 02 -4

aminophthalate + N2 + photon [25]

and as such, provides a measure of thephagocyte DOX capacity.

Estimating Pagocyte OpsoninReceptor Reserve: TheoryThe humoral-phagocyte axis of the inflam-mation response is essentially an informa-tion-effector system directed to the task ofkilling pathogenic microbes. Tissue injuryor infection elicits the synthesis and releaseof immunologic information in the form ofcytokines and other inflammatory media-tors. These mediators affect the expressionof membrane receptors as required foreffective phagocyte-endothelial contact,diapedesis and locomotion through theinterstitial space to the site of injury orinfection.

Expression of phagocyte opsonin, i.e.,complement and immunoglobulin, recep-tors increases with the level of exposure toa diverse range of inflammatory mediators,including complement-derived C5a (com-plement anaphylatoxin), cell-derivedplatelet activator factors (PAF), leuko-trienes, tumor necrosis factors (TNF),colony-stimulating factors (CSF), and cer-tain interleukins such as IL-8, as well asmicrobe-derived factors such as N-formyl-methionyl peptides. It should be empha-sized that opsonin receptors are rapidlyexpressed on exposure to mediators andthat this increased expression does notrequire neosynthesis or prolonged incuba-tion. Opsonin receptor expression can beincreased with little or no activation of res-piratory burst metabolism. However,opsonin-opsonin receptor ligation triggersrespiratory burst metabolism. Under prop-erly controlled conditions of testing, phago-cyte opsonin receptor expression can befunctionally linked to the accelerationphase of the respiratory burst and mea-sured as luminescence (42-44).

Phagocyte opsonin receptor expressionis dependent on exposure to inflammatorymediators,

R/P = k[P] [I] [26]where R/P is the opsonin receptor (R)expressed per phagocyte (P), k is the pro-portionality constant, [P] is the concentra-tion of phagocytes and [I] is theconcentration of inflammatory mediators

capable of increasing opsonin receptorexpression. [Imax] is a concentration ofinflammatory mediator sufficient to pro-duce maximum opsonin receptor expres-sion Rmax/P.

Phagocyte receptor ligation of opsonins,such as complement and/or immunoglobu-lin-coated microbes, activates respiratoryburst metabolism yielding oxygenatingagents (X),

dXldt = k[R/P] [P] [0] = k[R] [0]

where dXldt is the rate of oxygenatingagent generation, [0] is the concentrationof opsonin, and the other components areas previously described. If the conditions oftesting are set such that [0] is muchgreater than [R], the equation simplifies to,

dXldt = k[R]. [28]

Since dLIdt is a function of [X], it followsthat

dLIdt = k[R]. [29]

A commercially available luminescencesystem is now available for measurement ofbasal and stimulated phagocyte oxidase andoxidase-driven haloperoxidase activities.The system also measures both the circulat-ing opsonin receptor expression (CORE)and the maximum opsonin receptorexpression (MORE) per phagocyte. TheCORE value is obtained by testing amicrometer quantity of unmodified bloodunder conditions where [0] and [C] arenonlimiting; i.e.,

dLldt = k[R] = k[P][Iin vivo] [30]

The MORE value is obtained by simul-taneously testing an equivalent quantity ofthe same blood specimen with the samenonlimiting concentrations of0 and C butwith sufficient inflammatory mediator, i.e.,Imax, to ensure maximum opsonin receptorexpression but minimum azurophilicdegranulation and minimum direct activa-tion of respiratory burst metabolism. Theresulting luminescence response to 0 repre-sents maximum receptor activity,

dLm xldt = k[Rmax] = k[P][Imax] [31]

The luminescence responses L and Lmareflect the CORE and MORE, respec-tively, and can be presented as a ratio, theCORE/MORE ratio or inflammatoryindex (42-44). Low ratio values reflectminimum in vivo exposure to inflamma-

tory mediators. The ratio increases toreflect the level of in vivo exposure toinflammatory mediators. The ratio can alsobe presented in reciprocal form as the per-centage opsonin receptor reserve (%ORR)per phagocyte,

1-(L/L) x 100 = %ORR [32]

The %ORR decreases in proportion tothe degree of in vivo stimulation.

Estimating PhagocyteOpsonin Receptor Reserve:The ApplicationAnalysis ofPhagocyte OpsoReeptor ExpressionMaterials and Methods. Basal and stimu-lated phagocyte oxidase and oxidase-drivenhaloperoxidase activities were measuredwith the AXIS luminescence system andreagents (ExOxEmis Inc., San Antonio,TX). Both the CORE and the MORE perphagocyte were also measured using thissystem. The K EDTA-anticoagulatedwhole blood coleected for complete bloodcount with differential was kept at ambienttemperature (20-230C) and tested within2 hr of venipuncture. Just prior to mea-surement, 100 pl of whole blood wasadded to 9.9-ml blood-diluting medium(BDM, an AXIS reagent) for a 1:100 dilu-tion. The diluted blood was loaded intothe luminometer (Berthold LB953 AXISmodified, Wildbad, Germany) for auto-matic injection into prefabricated test tubescontaining the indicated coating of inflam-matory mediator or PMA. Tubes coatedwith PMA were used for opsonin receptor-independent measurement of oxidase andoxidase-driven haloperoxidase activities.

On initiation of measurement, theluminometer injected 600 pl of eitherluminol balanced salt solution (LBSS, anAXIS reagent) or dimethylbiacridinium(DBA++, i.e., lucigenin) balanced salt solu-tion (DBSS, an AXIS reagent), and 100 plof diluted blood, i.e., 1 pl equivalent ofwhole blood, into each tube. LBSS andDBSS contain non-rate-limiting quantitiesof luminol and DBA++ as the respectivechemiluminigenic substrate (C) to ensurethat the luminescence response reflects thequantity of oxygenating agent generated bythe activated leukocyte and not the avail-ability of C. All of the luminescence mea-surements were in triplicate and were takenover a 20-min interval (approximately 1measurement/1.5 min). The absolute poly-morphonuclear neutrophil leukocyte(PMN) count was used to calculate the

Volume 102, Supplement 10, December 1994 205

R. C. ALLEN

specific luminescence activity expressed astotal counts/PMN/20 min. Alternatively,specific activity can be expressed ascounts/phagocyte/20 min. The totalphagocyte count is the total leukocytecount minus the lymphocyte count.

Opsonin receptor-dependent oxidase-driven MPO activity was measured withLBSS as the C medium. Human comple-ment opsonized zymosan (hC-OpZ, anAXIS reagent) was used as the opsonin.Response to this opsonin was tested in theabsence and presence of sufficientinflammatory agent to produce maximumneutrophil opsonin receptor expression.The data presented in Table 3 wereobtained using uncoated tubes, recombi-nant human C5a-coated tubes (C5a, 20pmole tubes, an AXIS reagent) and PAF-coated tubes (PAF, 10 pmole tubes, anAXIS reagent) with 100 pl complement-opsonized zymosan (108 zymosan particlesper test).

Figure 1 presents the plot of lumines-cence velocity, dLldt, expressed as mega-counts per minute (CPMx 1 06) (theordinate) versus time in minutes (theabscissa) for both CORE (lower curve) andC5a-MORE (upper curve) responses to 0,hC-OpZ. The leukocyte count was 5100/p1with an absolute differential count of 3200segmented PMN, 205 monocytes, 51eosinophils and 1630 lymphocytes/pl. Eachmicroliter of blood tested contained 3200PMN and 3456 phagocytes. The %ORR iscalculated from the initial 10-min integralresponse. During this initial period, activa-tion is causally linked to opsonin receptorexpression. After this initial period, phago-cyte metabolic capacity begins to exert alimiting effect on the luminescenceresponse. The initial integral CORE (i.e.,L) response is 2.6 x 106 counts/10 min, andthe C5a-MORE (L max) response is13.0 x 106 counts/10 min. Therefore, theinflammatory index is 0.20 and the %ORRis 80. This %ORR value is within the nor-mal range of 77±10 (SD) for healthy sub-jects (n = 20). Table 3 presentsoxidase-dependent and oxidase-haloperoxi-dase-dependent (both opsonin receptordependent and independent activities) inte-gral 20-min specific luminescence responsesfor the same blood specimen. Imax primed,hC-OpZ-stimulated phagocytes typicallyshow peak dLldt by 15 min. Once peakdLldt is reached, phagocyte metaboliccapacity exerts a limiting effect. As such,the 20-min MORE response reflects meta-bolic capacity. The MORE responses areessentially the same with either C5a or PAFas immunologic modulator.

Table 3. Whole blood luminescence analysis.

C O or PMA Na azide L,medium immune primer stimulus inhibitor Cts/PMN/20 min Phagocyte function measured

LBSS None None None 13 Basal DOX activityLBSS C5a, 20 pmole None None 22 C5a-primed DOX activityLBSS PAF, 10 pmole None None 35 PAF-primed DOX activityLBSS None hC-OpZ None 2,596 CORE activityLBSS C5a, 20 pmole nC-OpZ None 12,354 C5a-MORE activityLBSS PAF, 10 pmole hC-OpZ None 11,385 PAF-MORE activityLBSS None PMA, 5 nmole None 1,844 PMA-stimulated DOX activityLBSS None None None 19 Basal RDOX activityLBSS None PMA, 10 pmole None 387 PMA-stimulated RDOX activity

Azide inhibition, %LBSS None None 5 nmole 15 -15LBSS C5a, 20 pmole None 5 nmole 20 9LBSS PAF, 10 pmole None 5 nmole 22 37LBSS None hC-OpZ 5 nmole 464 82LBSS C5a, 20 pmole hC-OpZ 5 nmole 2,426 80LBSS PAF, 10 pmole hC-OpZ 5 nmole 2,501 78LBSS None PMA, 5 nmole 5 nmole 370 80DBSS None None 5 nmole 21 -11DBSS None PMA, 10 pmole 5 nmole 423 -9

Reaction initiated by addition of 1 pi blood suspended in 99 p1 diluting medium to the prefabricated tubes as indi-cated. Each pl blood contained 3200 segmented PMS leukocytes, 1600 lymphocytes, 200 monocytes, and 50eosinophils. The tubes contained the indicated quantity of primer or PMA, plus 600 pl LBSS or DBSS and, whereindicated, 100 pl hC-OpZ. The blood was tested in triplicate measurements within 2 hr of venipuncture. AXISreagents were used throughout and measurements were taken with a Berthold LB953 luminometer.

3.0- * A24AU3rl/16+17+180 A24AU3rl/19+20+21

2.5-

= , 2.0-

a c 1.5~ -

C. a)C

1.0

15 20

Time, in minutes

Figure 1. The CORE and C5a-MORE luminescence responses of 1 liter blood with LBSS as C and hC-OpZ (1 08 parti-cles) as 0. Reaction was initiated by injection of diluted blood at time zero. All specimens were tested in triplicate.

Relatively low concentrations of azide(N;) inhibit haloperoxidase (37,45). Theselective inhibitory effects of sodium azide(5 nmole/test; 6 pM final) on the LBSSand DBSS luminescence responses ofPMN to 0 and PMA stimulation are pre-sented in the lower portion of Table 3. Theeffects of azide on the CORE and C5a-MORE raw luminescence responses arepresented in Figure 2. Although a finalconcentration of 6 ,iM of azide inhibitsluminescence by approximately 80%, the

%ORR is increased slightly from 80 to86%. This small increase may reflect agreater susceptibility of CORE activity toazide inhibition. As shown in Table 3,azide also inhibits PMA-stimulated LBSSluminescence by approximately 80%.

Azide does not inhibit DBSS lumines-cence in response to PMA (10 pmole/test).As shown in Table 3, azide produces amild augmentation that probably reflectsinhibition of MPO-mediated oxidationactivity which can interfere with DBA++

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PHAGOCYTE OXYGENATING ACTIVITY

3.0' * A24AU3rl/16+17+180 A24AU3rl/19+20+21

2.5

Ec 2.0-

U=

cu 1.5a,E

1.0

0.5

O 0- ----

0 5 10 15 20

Time, in minutes

Figure 2. The conditions were the same as described for Figure 1, except that 5 nmole of sodium azide wasincluded per test (6 pM azide final concentration) as a myeloperoxidase inhibitor.

* A24AU3r3/37+38+39O A24AU3r3/43+44+45

1.5

o 1.0

E

0.5

0)

0 5 10 15 20

Time, in minutes

Figure 3. The temporal luminescence responses of 1 pl blood with DBSS as C and PMA (10 pmole/test) as chemi-cal stimulus. The upper and lower curves depict the luminescence responses in the presence and absence ofsodium azide (5 nmole/test; 6 pM final), respectively.

reductive dioxygenation. The effect ofazide on the raw DBSS luminescenceresponses from PMA-stimulated blood isillustrated in Figure 3. The lower andupper curves depict the responses to PMA(10 pmole/test) in the absence and pres-ence of azide, respectively.

All of the luminescence data presentedwere obtained from less than 0.5 ml bloodand required less than 1 hr for setup, mea-surement, and analysis. Luminescence test-ing allows rapid, high sensitivity differentialanalysis of phagocyte oxygenation activi-ties. If the conditions of analysis are prop-erly controlled, opsonin receptor expressioncan also be measured, and since opsoninreceptor expression is in proportion to thedegree of inflammatory mediator exposure,analysis of the circulating phagocyte pro-vides a gauge of in vivo inflammation. TheAXIS system is ultimately designed for usein patient diagnosis and management(46,47). However, the system also hasbroad potential for assessing inflammationand antiinflammatory activity using in vivoand in vitro models (48).

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