0099-2399/97/2306-0366503.
00/0
JOURNALOF ENOODONTICS
Copyright © 1997 by The American Association of Endodontists
Detection of Human IL-2 in Normal and Inflamed
Dental Pulps
Cindy R. Rauschenberger, DDS, MS, Jeffrey C. Bailey, DDS, MS, and Cynthia J. Cootauco, MS, DDS
Irreversible pulpitis has been associated with an in-
crease in the number of pulpal T-cells. Interleukin-2
(IL-2) stimulates T-cell proliferation and signals ~ re-
lease of other proinflammatory mediators associated
with connective 1issue degradation. IL-2 has been sug-
gested to be a useful marker of pathologic inflamma-
tory activity in periodontal and systemic disease con-
di~ons. The purpose of this study was to analyze
normal and inflamed dental pulps for the presence of
immunoreacUve IL-2 (ilL-2).
Normal healthy pulpal tissue was obtained from
17 impacted third molars and inflamed samples
were obtained from 12 symptomatic carious mo-
lars clinically diagnosed with irreversible pulpitis.
Pulpal tissues were collected, prepared, and ana-
lyzed for histological status and ilL-2 concentra-
tion by a modified ELISA technique, ilL-2 was de-
tected in all vital pulpal tissues. A t-test revealed
significant differences in ilL-2 concentrations
when inflamed pulpal tissues were compared to
normal healthy samples (T = -2.75, p < 0.05).
These results suggest that ilL-2 may serve as a
marker of pathologic inflammatory activity in irre-
versible pulpitis.
Clinical pulp tests indirectly attempt to evaluate inflammation or
the pathologic effects of inflammation on the pulpal and surround-
ing periradicular tissues. However, the patient response and inter-
pretation of these tests may vary from day to day or from clinician
to clinician. Therefore, current clinical tests are incapable of de-
termining quantitatively and definitively the health status of pulpal
tissue.
Following insult to connective tissues throughout the body,
biologic molecules (i.e. inflammatory mediators) are released from
damaged cell membranes to recruit inflammatory cells into the area
of injury or bacterial penetration. Diagnostic assessment of these
biologic markers may improve the validity of predicting or treating
inflammatory pulpal disease. Thus it is important to determine the
concentrations of these molecules in healthy and diseased pulpal
tissues.
366
Printed in U.S.A.
VOL. 23, NO. 6, JUNE 1997
Cytokines are intercellular regulatory proteins that mediate mul-
tiple immunologic and nonimmunologic biological functions (1).
The term interleukin (IL) is used to describe a cytokine that is
responsible for intercellular communication between leukocytes.
The association or correlation of cytokines with systemic inflam-
matory disease or tissue pathosis has been well documented (2, 3,
4,5,6).
The normal biologic reaction of the dental pulp to injury is
inflammation and possibly a T-cell mediated immunologic re-
sponse. Investigators have evaluated the immunologic response of
the dental pulp to caries in both normal and inflamed conditions (7,
8, 9). When irreversibly inflamed pulpal tissues from carious teeth
were compared to normal samples, increased numbers of T-cells
were reported (10)+ These authors suggested that T-cells are re-
sponsible for the regulation of pulpal immunopathic changes under
carious lesions.
Interleukin-2 (IL-2) is a potent immunostimulantproduced and
secreted principally by helper T-cells (11). IL-2 stimulates colonal
expansion of the helper T-cell population plus it activates a mul-
titude of other cytokines that orchestrate the cell mediated immune
response in connective tissues (1). IL-2 may play a role in the
pathogenesis or progression of pathologic inflammatory disease
and it has been suggested to be a useful marker of disease activity
in systemic as well as periodontal disease conditions (6, 12).
Currently little is known about the concentration of IL-2 in the
human dental pulp. Because the connective tissues, etiologic fac-
tors, destructive inflammatory processes, and immunologic reac-
tions of pulpal disease are similar to those of periodontal disease,
it is plausible that IL-2 may also be used to signal the progression
of irreversible inflammatory pulpal conditions.
The endodontic literature is replete with articles that report a
lack of correlation between patient symptoms and histological
pulpal findings (13, 14, 15). However, these investigators did not
attempt to document the presence or upregulation of biologic
molecules responsible for inflammatory vascular changes and in-
flammatory cell recruitment to the affected tissue areas. Biologic
chemotactic molecules may be present before the actual histolog-
ical appearance of the inflammatory cells in injured pulpal tissues.
It is also possible that specific populations of T-cells and/or up-
regulation of their cellular product, interleukin-2, may be associ-
ated with clinical symptoms as well as inflammatory histological
changes in pulpal tissues.
The purpose of this study was to determine if IL-2 can be
detected in normal and inflamed pulpal tissues classified by clin-
ical symptoms and histological assessment.
Vol. 23, No. 6, June 1997
MATERIALS AND METHODS
Sample Collection
Health history reports were reviewed to determine that each
subject was healthy. A total of 40 patients agreed to participate in
this study. Written informed consent was obtained from each
subject after the nature of the procedure and possible discomforts
or risks had been explained fully. Twenty pulp samples were
obtained from patients with caries free, asymptomatic, soft tissue
impacted third molar teeth (i.e. normal or healthy pulpal tissues).
The clinical diagnoses of the teeth in the normal group were
confirmed with radiographs. The patients in this group were
asymptomatic and the experimental teeth exhibited no radio-
graphic evidence of caries or periapical pathosis. The teeth in the
normal group were extracted as part of each patient's treatment
plan.
Twenty inflamed pulpal samples were collected from patients
with carious molar teeth whose clinical symptoms were consistent
with that of irreversible pulpitis. A diagnosis of irreversible pul-
pitis was made from an evaluation of periapical radiographs, sub-
jective history, and a clinical examination. The clinical findings
included detection of caries with an explorer, a history of sharp
intermittent pain to thermal stimuli, a lingering painful response to
the application of a cold stimulus, no pain to percussion tests, and
a positive response to electric pulp testing (EPT). These teeth
exhibited deep coronal caries and no radiographic evidence of
periapical pathosis. The patients in this group elected extraction
rather than endodontic therapy.
Local anesthetic was administered and all experimental teeth
were extracted routinely without complications using elevators and
forceps. Immediately following extraction, a longitudinal groove
was placed in the crown of the tooth under water coolant with a
#557 carbide bur and high speed. The teeth were fractured by
twisting a #3 elevator in the prepared groove. Following tooth
fracture, the pulpal tissue was gently removed and sectioned lon-
gitudinally into equal halves with a Bard-Parker scalpel blade.
Approximately one-half of the recovered pulpal tissue was placed
in 4% paraformaldehyde for histological evaluation, while the
remainder was frozen at - 7 0 ° C until prepared for enzyme linked
immunosorbent assay (ELISA) procedures.
Histological Evaluation
All pulpal tissue samples fixed in 4% paraformaldehyde were
embedded in low temperature paraffin, sectioned at 6 txm, and
stained with hematyxalin and eosin (H & E). The H & E slides
were examined in a blind fashion by 2 investigators (J.B. and C.R.)
at 100 ×, 250 ×, and 400 × magnification and categorized as
follows: normal pulp, mildly inflamed pulp, moderately inflamed
pulp, and severely inflamed pulp according to criteria published
previously (16, 17).
Enzyme-Linked Immunosorbent Assay (ELISA)
Frozen pulpal samples were thawed for 30 min and weighed.
One hundred/xl of phosphate buffered saline (PBS), pH 7.0, was
added to each specimen and the tissue crushed (50 strokes against
the collection vial) with a small glass rod in an attempt to etute IL-2
from the pulpal tissues. The elutions were performed at 4°C over
Pulpal IL-2 Detection 367
a 30-min period with mixing before centrifugation for 2 min at
9880 g. Fifty/xl of the supernatant fluid was diluted 10 fold with
PBS before assay.
Standards, Human-IL-2 (Calbiochem, San Diego, CA), and the
eluted pulpal samples were applied to 96 well-microtiter plates in
quadruplicate and assayed according to the modified ELISA pro-
cedure as follows:
I) Forty /,zl of standard or sample was applied to XenobindT M
(Xenopore Inc.; Saddlebrook, N J) microtiter plates in a predeter-
mined manner and incubated at 37°C for 2 h. The XenobindT M
plastic microtiter plate contains an amide membrane in each well
to covalently bind protein in the applied standard or sample prep-
arations.
2) Three hundred thirty/xl of 1% bovine serum albumin (BSA)
in PBS was applied to the microtiter plates to completely fill each
well, and the plates were incubated at 37°C for 1 h. BSA was
applied in an attempt to block or prevent nonspecific binding of
reagents to unreacted sites in the microtiter plate well.
3) The plates were washed with 1% BSA and 5% Tween 20 in
PBS pH 7.0 (PBST) to eliminate unreacted material, and 50/xl of
1:1,000 rabbit anti-human IL-2 serum (Calbiochem; San Diego,
CA) was added to the plate for incubation at 37°C for 40 min. This
was followed by a wash in 1% BSA in 5% PBST.
4) Fifty/zl of 1 : 1,000 biotinylated goat anti-rabbit IgG (Calbio-
chem: San Diego, CA) serum was added to the microtiter plate and
it was incubated at 37°C for 40 min and washed.
5) Fifty/zl of 1: 1,000 avidin alkaline phosphatase (Calbiochem:
San Diego, CA) was added, and the plate again incubated at 37°C
for 40 rain and washed (avidin has an affinity for biotin; therefore,
it should bind to all biotinylated IgG molecules in the microtiter
well attaching alkaline phosphatase to each of these molecules).
6) Forty/xl of the primary substrate (NADP) was added to each
well and incubated at 37°C for 15 min.
7) One hundred ten /xl of the secondary substrate (NaPHO4
buffer, p-iodonitrotetrazolium violet, diaphorase, alcohol dehydro-
genase, and 95% ethyl alcohol) was added to each well to allow the
reaction with alkaline phosphatase to occur. This enzymatic reac-
tion produced a colored product (purple formazan) directly related
to the concentration of iIL-2 in each sample. The amount of color
production in each well was analyzed spectrophotometrically at a
wavelength of 492 nm.
8) Optical densities (OD) of the individual wells were obtained
spectrophotometrically (492 nm) 5 rain after the addition of the
secondary substrate. (The optical density reflects the absorbance of
the purple formazan product developed in each well at this period.)
Protein Concentration Calculations
An average OD was calculated for the respective wells of each
IL-2 standard and pulpal sample assayed in quadruplicate on the
microtiter plate. With the use of standard dilution concentrations
and ODs, a stand~d curve was prepared for each plate, and the
amount of iIL-2 (units/p,1) was calculated by linear regression
analysis for each pulpal sample.
The amount of ilL-2 units (/x) per mass of tissue specimen (mg)
was determined using the following formulas:
Pulp weight (mg)
a) 0.100 (ml) = Pulp concentration(mg/ml)
IL-2 concentration (/x/ml) ilL-2 (/z)
b) Pulp concentration (mg/ml) - pulp (mg)
368 Rauschenberger, et al. Journal of Endodontics

Statistical Analysis Mean ilL-2 Concentrations in

Pulpai Tissue
The 1L-2 data in ~/mg of tissue were statistically analyzed using
a t-test for the two groups classified by clinical diagnosis and using
(Clinical Classification)
an analysis of variance (ANOVA) for the groups classified by
2,000
histological assessment. An alpha level of 0.05 was selected for
statistical significance, and a post hoc Newman Keuls test was
used to determine the significance between groups as detected by 1,500
ANOVA.
u/mg t,000,
RESULTS
500,
Eight samples were lost during preparation for ELISA analysis
(3 normal and 5 clinically inflamed tissues), and 4 samples were
0,
lost during histological preparation (1 normal and 3 of the clini-
cally inflamed tissues). Inflamed Normal
A summary of the results of the experimental samples are listed FIG 1. Graphic representation of mean ilL-2 concentrations in pulpal
in Table 1. iIL-2 was detected in all vital pulpal tissues. Pulpal tissue according to clinical diagnostic categories (irreversibly in-
tissue #27 was clinically diagnosed as normal but histological flamed v/s normal pulpal tissues).
analysis confirmed it to be necrotic (lack of recognizable connec-
tive tissue architecture) and the iIL-2 concentration in sample #27 TABLE 2. Statistical analysis of ilL-2 data categorized
was below detectable limits of the ELISA procedure. according to clinical diagnosis,
The ELISA data for IL-2 were categorized according to clinical
t-test: ilL-2 / mg pulpal tissue
diagnosis and statistically analyzed using a t-test. Refer to graphic
representation data in Figure 1. The t-test revealed significant (CEnical diagnosis)
differences between iIL-2 concentrations in tbe clinically normal
and irreversibly inflamed pulpal tissues. The inflamed group dem-
Normal Inflamed
onstrated a mean of 1944.75 _+ 2050.97 units iIL-2/mg pulpal
tissue versus 541.29 +- 449.55 units iIL-2/mg for the normal pulpal Mean 541.29 1944.75
tissue group (Table 2).
Of the 20 samples collected for the clinically normal group, Std. Dev. 449.55 2050.97
histological analysis of the samples revealed 13 normal tissues, 1
t = -2.75, p<0.05

TABLE 1. Experimental data obtained for each sample

collected in the study. necrotic sample, and 3 mildly inflamed tissues. Three samples
Experimental data were lost as previously described during histological processing or
EL|SA preparation.
s~pte n t~th 0 ~,eight (~) Clinical /tlstoloficat 1L-2wm~
I 1 0018 normal nolmal 459
In the group consisting of 20 inflamed samples, 11 were con-
2
3
16
~2
0029
0 028
rmtmal
r,ormal
a
normal
97
firmed to be histologically inflamed, 1 was determined to be
4
5
14
16
0005
0O06
inflanl~
notnml
a
normld
b
613
histologically normal, 3 were too small for processing, and 5 were
6
7
30
14
OOO9
0003
inflamed
iPJ]amcd
moderate
modcral¢ h
b
lost during ELISA preparation. The 11 histologically inflamed
8
9
31
18
0(1(16
0.(~9
inllamed
inflamed
mild
stwcrc
1140
180
tissues were further categorized according to the extent of inflam-
J0
II
3
16
0.003
0Ol
inflanmd
inflamed
sea,ere
mild
2225
668
mation (16, 17). Five samples were classified as mildly inflamed,
12
13
17
3
0.005
0 (i(12
normal
ilLOamed
normal
/llO~mtc
510
h
3 as moderately inflamed, and 3 as severely inflamed.
14
15
2
30
0013
OOO3
rmrmal
inflamed
normal
moderate
b
1910
Figure 2 graphically represents pulpal iIL-2 concentrations ac-
16
17
31
32
0102
0.011
inflamed
normal
moderate
normal
b
198
cording to histological classification. ANOVA revealed that sig-
18
19
19
32
(l019
0011
inflamed
norn~l
norn~l
normal
105
470
nificant differences existed between the histological groups (F =
20
21
I
16
I) 1)15
0015
normal
normal
normal
normal
195
621
3.24; p < 0.05). A post hoc Newman Keuls test revealed that
22
23
30
M
0tK~6
0t~)9
itdlarrcd
iallamcd
moderate
severe
738
1258
significant differences existed between all histologically inflamed
24
25
2
I
(} C413
0Ol
inflamed
normal
a
mild
2905
556
tissue groups (i.e. mild, moderate and severe pulpal inflammation)
26
27
16
I
0.¢~5
0(105
intlamcd
normal
moderate
nocrolic
b
o
and the histologically normal pulpal tissue (F = 3.49; p < 0.05)
28
29
16
17
0005
O005
normal
inflamed
mild
a
1629
2676
(Table 3).
30 3O o1~5 inflamed moderate 2040
31 I5 OOO7 ildlanled mild I[10
32 32 0037 normal nornl~ 40tl
33 17 0025 nonlml normal b
34 1 0(g~) normal normal 1127 DISCUSSION
35 16 otto9 normal mild 1390
36 32 0024 nortaal normal 169
37 17 0016 normal nor~l 5(19
38 17 0019 normal normal 282 The present study has identified iIL-2 in all vital pulpal samples.
39 14 o OO3 inflamed mild 4935
4O 15 0012 inflated mild 7028 Histologically uninflamed pulps exhibited low concentrations of
iIL-2 irrespective of their clinical classification (normal or irre-
a *ampte Io ~t durm~tht~t,dogtcal pn,¢.e.,~xng
versibly inflamed). The highest concentrations of iIL-2 were de-
h wunph"h~,t during pro<e.~ing lut kLLS:
tected in mildly inflamed pulps followed by the moderate and
Vol. 23, No. 6, June 1997 Pulpal IL-2 Detection 369

Mean t1L-2 Concentrations in trations than those reported for healthy pulps (19). These sub-
Pulpal Tissue stances are chemotactic for inflammatory cells such as PMNs and
macrophages. It is possible that these extracellular biologic mol-
(Histological Classification) ecules or messengers may precede the histologic appearance of
2,500 inflammatory cells in irreversibly damaged tissues. Detecting these
messengers, differentiating their physiologic concentrations from
2,000 pathologic concentrations, and associating their abnormal concen-
trations with pathologic inflammatory pulpal changes may im-
1,500. prove the validity of endodontic diagnostic testing. The present
u/mg study is an attempt to document the presence and relative concen-
1,000. tration of one important lymphocyte cellular messenger, IL-2, in
normal and inflamed pulpal tissues, ilL-2 is found in significantly
500 higher concentrations in the irreversibly and histologically in-
flamed pulpal tissue samples as compared to their clinically and
0- histologically normal counterparts. However, further investigation
Normal Mild Mod Severe is needed regarding biologic products and events associated with
FIG 2, Graphic representation of mean ilL-2 concentrations in pulpal the pathogenesis of the irreversible pulpal disease process.
tissue according to histological classification of the experimental Some intrinsic problems associated with this study include the
pulpal tissue samples. small volume of pulpal tissue available for experimental evaluation
(sample volume was restricted due to experimental design and the
anatomic or physiologic variations between patients) and the in-
herent dynamics of the inflammatory process in the dental pulp.
TABLE 3. Statistical analysis of ilL-2 data categorized Due to the limited amount of recovered pulpal tissue, 12 of the
according to histological classification. 40 samples could not be completely analyzed. Four were lost
/IL-2 in Dental Pulp during histological processing and 8 were too small to be success-
ANOVA/Newmuu Keuls
(Histelegicd diagn~is) fully divided into two pieces and processed for both histological
Count Mean Std. Dev and ELISA analysis. The total volume of coronal pulp tissue
Normal 410.9 2759 decreases with age and in response to injury. Therefore, to increase
1562.7 7171
the sample populations in future studies, biochemical processing
Severe ~ 1023,0 techniques and experimental methods specifically designed for
minute sample volumes should be used.
SS df MS F P
Additionally, variability occurred in the histological appearance
Treattmerd 18833512 3 4572602.5 3 49 003
Error 43223084 24 1463144
of the inflamed pulpal samples classified by clinical findings. This
Total 62056596 27 experimental finding of histological variability is consistent with
the findings of several other investigators (13, 14, 16, 17). One
Normal Severe Mod. Mild explanation for this problem is that clinical pulpal inflammation is
a dynamic process, and the patients presenting for endodontic
Severe *
evaluation have varying thresholds for pain or other signs and
symptoms. Thus, the inflammatory process may have just begun in
some patients presenting for treatment and qualifying for experi-
• = Stoups siSn~cantly diderot from e~chother
mental evaluation. Other patients may be asymptomatic or able to
tolerate discomfort and pathologic inflammatory pulpal changes
severely inflamed tissues. Additionally, it has been demonstrated for extended periods of time before seeking care. Another group of
that ilL-2 concentrations are significantly higher in irreversibly patients may be asymptomatic yet exhibit pathologic inflammatory
inflamed pulpal tissues as compared to asymptomatic normal sam- changes when their pulpal tissues are subjected to experimental
ples. Therefore, it is plausible that detecting IL-2 in significant evaluation.
concentrations from pulpal tissue may reflect a pathologic pulpal In addition to these variables, the clinical tests used for deter-
condition or active state of inflammatory pulpal disease subsequent mining pulpal inflammation are subjectively performed and inter-
to extensive caries. preted by each individual dental practitioner. Therefore, individual
To date, the biologic pulpal responses to injury and the events bias and varying levels of practitioner expertise can allow discrep-
that control these processes are not completely understood. A ancies in the interpretation of traditional clinical diagnostic tests.
variety of clinical signs, symptoms, and test procedures continue to This variability in patient presentation, practitioner interpretation,
be used to evaluate the health of the dental pulp subsequent to and histological tissue condition is believed to be responsible for
injury. However, none of these clinical tools are truly capable of the large standard deviations observed in both experimental
determining definitively the pathologic status of this tissue (18). groups. Increasing the experimental sample size in future studies
The pulpal health status as determined by these methods is sub- may better distribute the data levels obtained for ilL-2 and decrease
jective and left open to interpretation by the clinician. An objective the standard deviation in both groups.
analysis of biologic products directly associated with pathologic From a biochemical perspective, proteins are physically altered
pulpal conditions could improve the validity of diagnostic testing or denatured to some extent by repeated freezing and thawing.
in endodontics. Freezing the pulpal tissues before ELISA preparation and assay
Recently, several biologic molecules have been identified in may denature some of the naturally occurring proteins in each
inflamed pulpal tissues and found to be present in greater concen- sample. Preparing fresh eluates for each assay eliminates this
370 Rauschenberger, et al.
experimental variable. However, the expense of the IL-2 standards,
antibodies, reagents, and technical support to perform individual
assays on fresh samples is prohibitive. All experimental samples
analyzed in this study were subjected to standardized collection,
storage, and processing procedures. Pulpal tissues were carefully
removed from the tooth and frozen immediately following extrac-
tion, thawed for 30 min, weighed, and crushed. The supernatant
fluids were then diluted 1:10 and frozen in 1 ml aliquots. Each
sample was thawed again just before performing the IL-2 ELISA
assay on multiple tissue samples (therefore, each sample was
subjected to an identical number of freeze/thaw cycles). If some
experimental protein was lost during the sample preparation phase
as a result of freeze/thawing the tissues, it was a standard factor for
all samples.
Another experimental concern relates to whether complete elu-
tion of IL-2 can be accomplished with the procedures used in this
study. The ability of the crushing procedure to completely liberate
IL-2 from the sample tissues is unknown. Therefore, it is probable
that the prepared supernatant fluid contains less IL-2 than the intact
pulpal tissue sample. However, care was taken to uniformly crush
the experimental tissues in PBS with 50 strokes by the same
investigator and a standard force over a finite period. With these
precautions, the amount of IL-2 released from the tissue by the
standardized preparation technique is believed to be uniform for all
experimental samples.
A concern regarding tissue homogeneity relates to whether the
same constituents exist in each half of the pulpal tissue used for
ELISA and for histological analysis. This is not as much of a
problem for the samples in the normal group as long as thry are
split longitudinally. The number and types of cellular and connec-
tive tissue elements should be very similar in both portions. The
inflamed samples, however, may not exhibit uniform homogeneity.
According to experimental design inflamed pulpal tissue is mac-
roscopically divided into two distinct pieces (one for ELISA and
the other for histological assessment). With random sectioning of
these experimental samples, it is possible that one half of the tissue
could contain the entire inflamed area. Thus, one tissue section
would include high concentrations of IL-2 and other inflammatory
molecules or cells, while the adjacent tissue portion would exhibit
very little evidence of inflammation. In an effort to avoid this
experimental discrepancy, special care was taken to divide the
inflamed samples immediately subjacent to the carious area to
include macroscopically visible inflamed tissues in both collection
containers. A suggestion to further control and document this
experimental variable in future work would be to obtain serial
sections from the paraffin embedded tissues. These additional
tissue sections could be immunobistochemically analyzed for the
presence ilL-2.
This study provides relevant information concerning the patho-
genesis of pulpal inflammatory disease. The highest concentrations
of ilL-2 were detected in the mildly inflamed pulps, followed by
the moderate and the severely inflamed tissues. Thus, during the
early or mild phases of histological inflammation, ilL-2 concen-
trations are at their greatest. Perhaps this is related to the pulp's
immunologic attempts at repair by stimulating the expansion of the
helper T-cell population. To confirm this hypothesis, it would be
very helpful to determine the histological appearance and concen-
tration of ilL-2 in reversible pulpal inflammation and contrast these
findings to those present in irreversible pulpal inflammation. As
Journal of Endodontics
irreversible pulpal inflammation progressed histologically, the
ilL-2 concentration decreased (Fig. 2). It may be speculated that
the decrease in ilL-2 represents a point where the tissue is in the
late phases of irreversible inflammation and is progressing toward
total histological tissue necrosis. This hypothesis requires further
investigation.
In conclusion, immunoreactive ilL-2 can be detected in vital
human pulpal tissues. Furthermore, inflamed pulpal tissues con-
tained significantly more ilL-2 than normal human pulps. These
results suggest that iIL-2 may be an objective marker for deter-
mining the extent of pulpal inflammation associated with irrevers-
ible pulpitis.
This research was supported by a grant from the Research and Education
Foundation of the American Association of Endodontists.
Dr. Rauschenberger is a former assistant professor and Program Director,
Baltimore College of Dental Surgery and currently in private practice, Naper-
ville, IL. Dr. Bailey is a former endodontic resident currently in private practice,
Virginia Beach, VA. Dr. Cootauco is a former endodontic resident currently in
private practice in Baltimore, MD. Address requests for reprints to Cindy R.
Rauschenberger, DDS, MS, Associates for Endodontics, 720 S. Brom, Suite
102, Naperville, IL.
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