Ž .Mutation Research 428 1999 109–114www.elsevier.comrlocatermolmut

Community address: www.elsevier.comrlocatermutres

Evaluation of toxic assault on the immune system

Richard Hong )

Genetic Toxicology Laboratory, UniÕersity of Vermont Medical School, Burlington, VT 05401, USA

Received 29 November 1998; accepted 19 February 1999

Abstract

As a consequence of increased industrialization, human exposure to environmental toxins is growing daily. The effects ofthis exposure on the immune system is potentially a major health problem. Studies following human exposure have notunambiguously demonstrated a biologically significant weakening of the immune system, primarily because tests whichshow a high correlation with disease susceptibility have not been utilized. Such assessment of the immune system isparticularly important in developing countries where regulation of environmental contamination may be less vigorous. At thesame time, the ability to perform screening of large population in this setting is impaired. Measurement of the antibodyresponse to an antigen used for routine immunizations provides a simple but very informative assessment and is particularlywell suited as a minimalist approach to immune system assessment in developing countries. q 1999 Elsevier Science B.V.All rights reserved.

Keywords: Immunotoxicology; Immune system diagnosis; Biologic marker

1. Introduction

As the world becomes more and more industrial-ized, we are exposed to more and more agents whichcan have deleterious health effects. In the UnitedStates alone, over 2000 different chemicals at 1300

w xsites were identified as potential toxins 1 . Theimmune system is a potential target for such effects.A number of studies show toxic effects on theimmune system of numerous chemicals used in agri-culture and manufacturing, as well as medicationsemployed in general medicine which are not used toobtain immunosuppression. In addition, ultraviolet

) Tel.: q1-802-863-5716; Fax: q1-802-656-8333; E-mail:rhong@salus.med.uvm.edu

Žray exposure can modulate the immune system Ta-.ble 1 .

Exposure of an individual to an environmentaltoxin can result in a hypersensitivity reaction, au-toimmunity or loss of the defense capability of the

Ž .immune system immunodeficiency . Dose effectshave been shown and mechanisms defined in animalstudies. In humans, hypersensitivity reactions to anumber of chemicals are well-documented. How-ever, the issue of immune deficiency resulting fromenvironmental exposure remains controversial. Theprimary reason for this confusion is due to themethods by which the deficiency state is defined.The immune system has all too often been character-ized by the most convenient, but not the most infor-mative tests. Some of the more discriminating testsare labor-intensive, which compromise their applica-

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S1383-5742 99 00038-1

( )R. HongrMutation Research 428 1999 109–114110

Table 1Chemicals reported to show immunomodulation

a aChemical class In humans In animals

Pesticides 0r8 8r8Organochlorides 2r7 7r7Organophosphates 1r11 11r11Carbamates and thiocarbamates 0r8 8r8Pyrethoids 0r3 3r3Metals 3r4 4r4Metallic salts, organometals 1r8 7r8Solvents 4r5 5r5Halogenated aromatic hydrocarbons 4r4 4r4Hormones 2r2 1r2Gases 2r2 2r2Polycyclic aromatic hydrocarbons 0r4 4r4Mycotoxins 0r6 6r6Addictive substances 4r6 5r6Anti-inflammatory agents 2r3 1r3Antibiotics and antineoplastics 5r5 0r5Antifungals 1r1 0r1

a Numerator—number of chemicals with immunomodulating ef-fect; denominator—number of chemicals tested.

w xTable 2 adapted from Ref. 1 ; data based on in vivo studies.

bility for large surveys, further discouraging theiruse. I believe that the three most common pitfalls inthe assessment of the immune system are:1. Failure to appreciate that individual variation of

measured values is great;2. Failure to appreciate that overlap of values from

diseased populations with normal populations israre;

3. A belief that more is better.I would like to propose a schema for informative

assessment of the immune system which will yieldinformation as to the ability of the immune system toprovide protection for an individual. My focus is onwhether the observed change can lead to increasedsusceptibility to serious infections. This more restric-tive view does not address the issue of whether theenvironmental exposure has a subtle or mild effectupon immunity. Such a distinction is important ifone wishes to define an agent as completely benignor set the allowable exposure limit for no observable

Ž .adverse effect NOAEL . For more extensive treat-ments of testing, the reader is referred to the reportsof several committees which have considered these

w xissues in detail 2–4 .

2. Basic principles

I will consider only briefly the issues of hyper-sensitivity and autoimmunity. These complicationsare generally evident from a careful history and thetemporal and geographical association of the patientand the agent. Skin test challenges and measurementof specific IgE antibodies to the putative cause con-firm the relationship of hypersensitivity to the expo-sure. The less direct association of autoimmunitysymptoms with the agent makes the cause and effectrelationship more difficult to prove. Here, carefulepidemiological studies are necessary.

Protective immunity is provided by the elementsof the immune system, viz. the T and B lympho-cytes, macrophages, natural killer cells, various cel-lular secretions known as cytokines, and an enzymecascade known as the complement system. It ispossible that one effect of an environmental toxin isto interfere with either the elaboration or action ofcytokines or complement, but the evaluation of thosepossibilities is beyond the scope of this presentation.This discussion, then, is restricted to evaluation ofthe cellular components noted above and the majorsecretions of the B lymphocytes, the immuno-globulins.

To put the measurements of these componentsinto the appropriate perspective, it is important tonote that much redundancy is built into the immunesystem. If one plots the range of values for a givenimmune system assessment, one can see the breadthof values encompassed. Further, when that range iscompared with the range observed in patients withcongenital immune deficiency disorders, there is es-

Ž .sentially no overlap see Fig. 1 . From this perspec-tive, it is difficult to see how a reduction of anyimmune system measurement of even as much as 50to 75% of the mean values can be biologicallysignificant, even though the absolute value appearsquite small in comparison. The important thing toemphasize is that individuals with such values areimmunologically just as intact as any other.

In biology, the ‘gray’ area in between the rangesof those with overt life-threatening disease and thenormal asymptomatic population is difficult to as-sess. From my personal clinical experience, individu-als in whom such values are found are generally ofthree types.

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Ž 3.Fig. 1. Plot of absolute CD q T cells rcm in 35 patients with primary immunodeficiency disease and marked deficiency of the T cell4

system. The values are those at the time of diagnosis, prior to any therapy. The patients ranged in age from 10 days to 3 and 7r12 years; 32were male and 3 were female. All required transplantation for cure or died of infectious complications. Note that nearly all values fall belowthe lowest limit of the normal range which is partially shown by the shaded area. Normal values extend even above the shaded area. Onlyone patient had values which extended into this area.

Ž .1 Young infants or toddlers who are assessed forŽ‘increased frequency’ although by actual count, the

.number usually is not outside the norm of upperrespiratory infections will frequently have values of

Ž .immunoglobulins usually IgG which are slightlybelow the range of normal. Such children never haveserious life-threatening infections, their frequency ofinfections diminishes and the values of immuno-globulins rise into the normal range.

Ž .2 Individuals with a congenital immunodefi-Žciency who have had therapy bone marrow or thy-

.mus transplant to correct the deficiency, but thenumerical value of their immune system componentsdoes not attain normal values. Interestingly, suchindividuals are usually completely normal from thestandpoint of their immune systems and they do notsuffer from any infectious susceptibility.

Ž .3 Individuals who have had a bone marrowtransplant for cancer and received very high doses ofchemotherapy for the malignancy and in addition,some myeloablative treatment to permit engraftment.They may or may not have symptoms of immunode-ficiency which is often correlated with the presenceof a chronic process, graft-vs.-host disease, whichhas a profound immunosuppressive action. If the

Ž .recipient is young less than 35–40 years old , thelower values are less likely to be associated with

w xsymptoms of a deficiency state 5 .Most individuals with ‘gray zone’ values come

from groups 1 and 2. Individuals in group 3 have hadvery severe attacks upon their immune system byvirtue of the chemotherapy and radiation used, com-bined with the effects of the malignancy itself aswell as complications such as graft-vs.-host disease.Thus, in the absence of the horrendous decimation ofthe immune system wrought by the ablative regi-mens required in bone marrow transplantation usedin association with powerful anti-cancer chemo-therapy, gray zone values should usually be treatedas outliers of the normal range. However, resolutionof this ambiguity can be accomplished, as will bediscussed in a later section.

Computer technology has increased the capacityof medical analysis logarithmically. In the area ofcellular morphology, we are no longer restricted tointerpretations of characteristics detectable by mi-croscopy. In the time that it takes to perform adifferential count on 100 white cells using a lightmicroscope, a flow cytometer will have examinedthousands of cells and characterized them unequivo-

( )R. HongrMutation Research 428 1999 109–114112

cally using multiple surface markers, as well as sizeand composition characteristics. Originally, our dis-crimination of lymphocytes stopped at whether theywere small, medium or large, whether the nucleuswas condensed or open and the cytoplasm granularor not. Today, there are over 100 surface markersdefined for mononuclear cells which not only dividethem into subsets, but indicate states of activation,impending death and other functions. These assayscan be performed on hundreds of samples in a usualworking day. Thus, from a few milliliters of blood,an acute overload of information can easily result.The ready acquisition of this data tempts the investi-gator to accumulate more and more data. However,the biological usefulness of this information has notbeen fully defined.

I will present my view on what is the mostinformative approach for the assessment of the im-mune system from the perspective of studies whichare feasible in large field studies, particularly indeveloping countries.

3. Analysis of the immune system

Analysis of the immune system can be eitherquantitative or qualitative. Quantitative measure-ments provide cellular numbers or mass values ofimmunoglobulins. The availability of numerousreagents, combined with the ease of analysis pro-vided by flow cytometry, makes measurement of

lymphocyte subsets simple and accurate. Althoughover 100 surface markers can be detected, for routineclinical use, enumeration of four subsets is sufficient.It is adequate to determine the number of helper Tcells, cytotoxicrsuppressor T cells, B cells and natu-

Ž . Ž .ral killer NK cells Table 2 . Immunoglobulins aremeasured as major isotypes, IgG, IgM and IgAŽ .mgrdl , most commonly by nephelometry which,like lymphocyte enumeration, is automated and suit-able for handling many samples per day.

Obviously, if a given component of the immunesystem is absent, there can be no protection from thatcell line or its products. However, this situation isseldom seen. As mentioned previously, the range ofvalues encompassed by disease populations is usu-ally easily separated from the range of normals.Evaluation of immunoglobulin levels is further com-plicated by the fact that they are age-dependent, sothat a given value must be compared to its age-re-lated control. In general, values which fall in thegray zone are usually of no clinical significance;however, this ambiguity can be resolved by qualita-tive or functional analyses.

A functional analysis mimics the response whichthe immune system makes following antigen expo-sure. Following this event, the normal immune sys-tem increases the number of lymphocytes which bearthe receptor specific for the immunizing antigen. The

Ž .T cells of both CD qCD q helper and CD q3 4 3Ž .CD q killerrsuppressor phenotypes increase in8

number. The enlarged CD qCD qpopulation helps3 4

Table 2Lymphocyte surface markers

CD number Cell lineage Comment

Usual clinical profileCD T cell Part of the TCRrCD complex3 3

CD T cell Helper T cells; bind to HLA Class II4

CD T cell and NK cell Cytotoxic T cells; bind to HLA Class I8

CD NK, granulocytes, macrophages Fc receptor for IgG16

CD NK Used simultaneously with CD to measure NK56 16

CD B cell Mature B cells19

Less commonly usedCD RO Leukocyte Denotes the memory T cell45

Ž .CD RA Leukocyte Denotes naive unstimulated T cell45

CD Activated cell Measures activation; used as an early test of response to a stimulus69

Use of multiple surface markers is more precise—mature helper T cell: CD qCD qCD y ; mature killer T cell: CD qCD yCD q ;3 4 8 3 4 8

natural killer cell: CD qCD q .16 56

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the CD qrIgMqB cells which share the same19

antigen receptor to multiply and differentiate. As theB cell population increases, its secretion of IgMincreases the serum level to measurable amounts ofthe specific antibody. As the B cells divide further,class switching occurs and IgG and IgA are synthe-sized. Functional analyses measure these events fol-lowing an in vivo or in vitro antigen exposure.

The response of the immune system requires anincrease in cell number as the initial event. There-fore, proliferation is a reliable and sensitive indicatorof an appropriate response, and is conveniently mea-sured in vitro by tritium incorporation. T lympho-

Žcytes will proliferate nonspecifically i.e., cells with.many different T cell receptors respond when stimu-

lated by mitogens. This response means that surfacereceptors can be triggered to transduce signals inter-nally leading to the subsequent events that result incell division. However, the hallmark of an immuneresponse is its clonal or oligoclonal nature, i.e., theproliferating cells all share the same T cell receptorŽ . Ž .TCR or only a few five or six TCRs respond. Amore specific, but still not clonal or oligoclonalresponse comes from stimulation with alloantigenŽthe transplantation antigen of a nonrelated individ-

.ual . The inability to respond when stimulated eitherby mitogen or alloantigen implies a profound defectin the immune system. However, the converse is nottrue. Many immunodeficiency disorders do not arisefrom a defect which is reflected by such a total lackof responsitivity, but are still fatal. In other words,the stimulation by mitogen or alloantigen is so strongthat some degree of cellular recruitment and prolifer-ation can be induced even though the immune sys-tem is so compromised that it cannot respond tospecific antigen. However, it is only the ability torespond to a specific antigen which can be correlated

w xwith the ability to resist infection 6 .In a similar vein, for B cells, the most clinically

relevant test is the response to an antigen. In theclinic, one can employ an antigen which is ordinarilyused for routine immunizations and measure theantibody response following vaccination. If one

Žchooses an antigen which requires T cell help pro-.teins, carbohydrates , the antibody response con-

comitantly assesses a major T cell function as well.T cell function can also be evaluated by delayed

hypersensitivity skin tests, where antigen derived

from common bacteria, viruses, or fungi to whichindividuals are likely to have been exposed is in-jected intradermally and erythema and indurationmeasured 48 h later. This test also measures theability of the T cells to produce an inflammatoryreaction which is an important part of the mechanism

Žby which T cells contain infectious agents the gran-.uloma reaction . The problem with delayed hyper-

sensitivity skin tests is twofold. They are not reliablein young infants who may not have had sufficientexposure and they are technically demanding. Theantigens must be carefully injected and the readingsmade by experienced individuals. This technique isnot particularly well-suited to large mass screeningas usually performed, but when conducted by skilledinvestigators, informative studies can be accom-

w xplished 7 .

4. An approach

In consideration of the above, it has been sug-gested that a single, extremely simple but highlyinformative assessment of the immune system couldbe accomplished by injecting the population with anantigen commonly employed for routine immuniza-tions and measuring the antibody response. Thiswould have the beneficial action of providing aroutine immunization as well as testing both the Tand B cell components of the immune system. Whileit is possible that in a very unusual situation abilityto support B cell help by helper T cells could beretained while other T cell functions were lost, itseems quite unlikely. The serum sample for measur-ing the antibody response could also be used formeasurements of other physiological parameters asdesired by the investigators. Samples could be pro-cessed quickly in the field with minimal equipmentand sent to an appropriate central facility for analy-sis.

In a situation where more resources are available,one could add to this analysis, cellular phenotypedetermination and serum immunoglobulin levels ofthe major Ig classes, IgG, IgA and IgM. However,no matter what the composition of the final protocolis, it is important to emphasize that functional testsmust be included, which means that the response ofthe cells to some antigens needs be assayed.

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