Immune function in acute stress

A.B. Segala, S. Brunob and W.C.N. Forteb

a
National Research Council (CNPq) fellowship. bImmunology Section of Santa Casa Medical School and
Hospital, São Paulo, Brazil.

components in students participating in the present

ABSTRACT
study. In conditions of basal chronic stress, acute
The aim of this study was to evaluate immune stress may cause alterations in immune function.
function in acute stress in medical students before
academic examinations. Key words: Acute Stress, Immune Response, Im-
Twenty-five medical students were selected be- munoglobulins and complement
cause they presented intense acute stress, evaluat-
ed by the presence of the following classic signs:
cold hands, intense sudoresis in the extremities,
generalized sudoresis, paleness, tachycardia, con-
fused reasoning, nervous irritability, diarrhea, and INTRODUCTION
sleep disorders in the hours preceding the examina-
tion (agitated sleep, insomnia). The concept that stress can affect one’s health is
Methods: Immediately before the examination, not new, having been reported for centuries, espe-
peripheral blood was collected from the 25 students cially in cases of the chronic form. However, only re-
presenting acute stress to analyze T and B cells, cently have researchers been able to carry out a
CD4+ and CD8+ cells, immunoglobulins, and C3 and more detailed study of this fact, with suggested in-
C4 complement components, as well as phagocytic terrelation between the immune, nervous, and en-
activity in neutrophils and monocytes. These investi- docrine systems1-3. Thus, situations that cause an in-
gations were repeated in the same students in situa- crease in mental stress, especially if chronic, provoke
tions free of acute stress. The results of the two nervous and hormonal alterations that culminate with
samples collected from each student were com- the depletion of the immune system1-4.
pared. In the literature, there are few studies concerning
Results: The means and standard deviations immunocompetence and acute stress, and those
showed no significant differences for any of the pa- that do exist are mainly in experimental models. In
rameters analyzed (p ⱖ 0.01). rats, a reduction in the proliferation of thymus-de-
Conclusion: We conclude that acute stress did not pendent lymphocytes (T) in the lymph nodes and
cause changes in the lymphocyte subpopulations, spleen was observed after immunization of the mod-
phagocytic activity of neutrophils and monocytes, els when these were immediately subjected to
serum immunoglobulins, or C3 and C4 complement repetitive electric shocks 5,6. In yet another study,
with rats submitted to the same process, an accen-
tuated depression in the activity of peripheral natural
Correspondence: killer (NK) cells was observed 7. Swine models with
aggressive behavior presented a depression in the
Wilma Carvalho Neves Forte
Alameda Barros 399, Apto. 162 - Higienópolis cellular and humoral response 8 and those that were
São Paulo – São Paulo – Brasil submitted to stress for 72 hours presented depres-
CEP 01232-001 sion in the lymphocytic and mitogenic response 9.
E-mail: [email protected] Rats submitted to stress provoked by exposure to
low temperatures presented a reduction in corticos-

Allergol et Immunopathol 2006;34(4):136-40

 Segal AB, et al.—IMMUNE FUNCTION IN ACUTE STRESS 137

teroids, thyroidal hormones, and bursa-equivalent Evaluation of lymphocytes was done by separa-
(B)10 lymphocyte levels. Researchers have proposed tion of these cells using the Ficoll-Hypaque gradient
the possible existence of an immune-endocrine-ner- and posterior incubation with monoclonal antibodies
vous axis in mice11 and in birds12. CD19 (B lymphocytes), CD3 (T lymphocytes), CD4
Research about acute stress and immune re- (T auxiliary) and CD8 (T cytotoxic). All of the tests
sponse in humans has been done mainly in individu- were done in duplicate form.
als with specific diseases 7. It has been demonstrated In the study of phagocytosis by mononucleated
that patients who underwent post-traumatic stress cells, the following procedure was used: separation
present a reduction in the function of peripheral of 5x10 6 cells and incubation in three tests tubes,
monocytes13. Patients submitted to abdominal surg- one containing 10 8 particles of Zymosan (Zy) as the
eries for cholecystectomy when compared to other control, one containing Zy and homologous serum
patients submitted to the same surgery, though (HS), and one with Zy and autologous serum (SA).
through laparoscopy, presented reduction in the tu- Subsequently, the number of cells that phagocytized
mor necrosis factor (TNF) and in the release of su- three or more particles of Zy in a fixed number of
peroxide anions by monocytes14. Patients with coro- 200 monocytes was determined. All tests were done
nary diseases, when submitted to angioplasty after in duplicate form.
acute stress presented activation of the hypothala- For the evaluation of neutrophils, the spontaneous
mus-hypophysis-adrenal axis, accompanied by a re- and stimulated nitro blue tetrazolium test was done
duction in serum interleukins 1 and 2 (IL 1 and IL2)15. using bacterial lipopolysaccharides, obtained from
After the observation that some students present- heparanized blood and using 0.1 % buffered NBT. All
ed emotional alterations during exams, coinciding tests were done in duplicate form.
with a sequence of a greater incidence of infections, The immunoglobulin and complement component
like the common cold, flu, tonsillitis, and pharingitis, values were obtained using the Mancini method,
we proposed to investigate the immune response of based on the diameter of the precipitation halo,
these students, after the research protocol was ap- which was correlated with the standardized values
proved by the Institution’s Ethics Committee and in- for each batch used of the radial immunodiffusion
formed consent was obtained from each student. plaque, with readings performed after a 48 hour pe-
riod for IgG and 72 hours for IgM and IgA.
Statistical analysis of this study was done using
PURPOSE the Student-t test. For this, the Microsoft Excel
2000 software from the Microsoft Corporation® was
The aim of this study is to analyze the levels of T used.
and B lymphocytes, CD4 and CD8 positive cells,
phagocytosis by monocytes, assess the serum lev-
els of IgG, IgM, IgA and of the C3 and C4 comple- RESULTS
ment components, and use the nitro blue tetrazolium
test for neutrophils, in students presenting signs and Of the twenty-five students were chosen, since
symptoms characteristic of acute stress, and com- the reference value of 60 % or more for the presence
paring the exam results with the same values ob- of signs and symptoms proposed in the question-
tained from the same individuals in conditions free of naire. The students were selected to participate in
acute stress. this study, 64 % were women and 34 % were men,
and the mean age was of 20.8 (± 2.07) years. The ob-
tained results are shown in figure 1 and in table I.
METHODS

Twenty-five students were chosen with signs and DISCUSSION

symptoms of cold hands, intense sudoresis in the ex-
tremities, generalized sudoresis, pale skin, tachycar- Signs and symptoms of cold hands, intense su-
dia, confused reasoning, nervous irritability, diarrhea, doresis in the extremities, generalized sudoresis,
and sleep disorders in the hours prior to the exam (ag- pale skin, tachycardia, confused reasoning, nervous
itated sleep, insomnia). All of the blood samples were irritability, diarrhea, sleep disorders in the hours prior
obtained in the morning period. The exams were re- to the exam (agitated sleep, insomnia) presented by
peated for the same students in situations free of the students in this study are considered classic of
acute stress. The results of the two samples collect- acute stress. Though other students reported stress
ed from each individual were compared. in the mentioned situation, twenty-five students

Allergol et Immunopathol 2006;34(4):136-40

138 Segal AB, et al.—IMMUNE FUNCTION IN ACUTE STRESS

100%
Sim
90%
Näo
80%

70%

60%

50%

40%

30%

20%

40%

0%
Cold hands

Intense sudoresis

Generalized

Pale skin

Tachycardia

Dizziness

Dyspnea

reasoning

irritability

Irritability

Agited sleep

Hipersonia

Insomnia
Confused
in the extremities

Nervous
sudoresis

Figure 1.—Results of the questionnaire done prior to blood sample collection of the selected twenty-five students that presented stress prior to academic
medical exams.

were chosen, since the reference value of 60 % or preserved and because there was no difference be-
more was chosen for the presence of signs and tween the 2nd and 3rd test, we can put aside the
symptoms proposed in the questionnaire, with the question of the functionality of these cells as to the
goal of obtaining more objective values of acute phagocytosis digestion phase 18-22. Nitro blue tetra-
stress, though there is no need of division for this zolium (NBT) demonstrates that neutrophils are liber-
consideration. On the other hand, the applied ques- ating electrons because they are captured by the sol-
tionnaire demonstrated the exacerbation of the sym- uble NBT dye, reducing it to formazan. This particle
pathetic autonomous nervous system during this has a dark blue color and deposits itself on the cyto-
state of stress, having different combinations of the plasm of the neutrophils, indicating that the digestion
manifestations in all of the patients analyzed, proving phase is preserved. The stimulated NBT was used to
that which was compiled by several studies in the reinforce this test, due to the fact that the latex used
1980’s1-3. can double or even triple the values obtained by the
During analysis of the laboratory exams used for spontaneous test classically used for immunological
lymphocyte subpopulation counting using the mono- investigation 21. Immunoglobulin and C3 and C4 com-
clonal antibodies CD19 (B lymphocytes), CD3 (T lym- plement component serum value assessment was
phocytes), CD4 (auxiliary lymphocytes), CD8 (cyto- done using simple radial immunodiffusion, accord-
toxic T), we verified that these are considered ing to the classic methods developed since Mancini.
reliable for the evaluation of lymphocyte popula- We did not observe a statistically significant dif-
tions16,17. For laboratory complementation, phagocy- ference between the results of the samples collect-
tosis by monocytes in three separate tests was done ed during the period of acute stress and of the con-
using living cells: the first aiming to analyze the via- trol obtained for each student. It is possible that the
bility of these cells, in other words, to determine if stress presented by some students facing exams
these remain alive during the experiment (CO), and for graduation, though many times associated to in-
the second and third aiming to analyze the function- fectious processes, is not as intense as the stress
ality of the monocytes, with the second test contain- induced in experimental models, in which immune al-
ing homologous serum (HSM) in which zymosan that terations were observed 5-10. It is even probable that
is activated by the complement system of a pool of the acute stress presented by patients prior to surg-
normal human serum activates component C3b that eries may affect the immune response even more.
joins to a specific receptor of these cells, allowing The literature presents few references to acute
phagocytosis; the third test is done with serum of stress in humans free of specific diseases, and fur-
the patient (SA), which also acts on the complement thermore it is a controversial subject in respect to
allowing phagocytosis to occur. With the first test this.

Allergol et Immunopathol 2006;34(4):136-40

 Segal AB, et al.—IMMUNE FUNCTION IN ACUTE STRESS 139

Table I Recent studies with patient samples more similar

Means and standard deviations of the lymphocyte to ours corroborate our results. In a study done in
subpopulation count, phagocytosis by monocytes, Chile in which 42 medical students were evaluated in
nitro blue tetrazolium test for neutrophils, serum stressful situations few minutes prior to final exams,
immunoglobins (A, M, G), serum C3 and C4 complement the researchers did not observe alterations in the
components in twenty-five medical school students, lymphocyte subpopulation count, in spite of elevated
under situations of acute stress and in situations free
of acute stress serum levels of cortisol and the reduction of these
cells in relation to mitogens 4. Likewise, a study done
Situation Mean ± SD P with professors that maintain daily chronic stress be-
cause of the work entailed, and that were submitted
B Lymphocyte Stress 567.2 238.87 0.60 to acute stress through the undertaking of resolving
Control 537.01 155.56 enigmas with incomplete solutions, in a restricted
T Lymphocyte Stress 2545.44 953.64 0.78 period of time, followed by the explanation of this to
Control 2520.29 723.55 the researchers, it was observed that the biological
CD4 + Stress 1468.76 578.93 0.98 variation of the organism is intimately related to indi-
Control 1464.27 435.16 vidual characteristics, varying with the level of chron-
ic basal stress and not to the level of acute stress 24.
CD8 + Stress 990.56 388.03 0.67
Control 1032.70 304.96 This can be correlated with results of our study when
we observe the individual results, due to the fact that
Ratio Stress 1.52 0.28 0.28
the students who participated in our study could
CD4/CD8 Control 1.44 0.25
have maintained an increased basal stress through-
Phagocytosis CO Stress 26 6.15 0.39 out their entire college education, presenting only
Control 28.06 7.91 non-significant immunologic alterations at the mo-
Phagocytosis HS Stress 67 5.92 0.76 ment of acute stress.
Control 66.39 5.96 We conclude that immunologic evaluation of med-
Phagocytosis AS Stress 71.78 5.11 0.47 ical school students, selected because they present-
Control 70.39 6.14 ed clinical signs and symptoms of acute stress pre-
NBT-EP Stress 7.4 1.39 0.33 ceding academic exams, did not demonstrate an
Control 7 1.17 alteration in the lymphocyte subpopulation count, of
the phagocytotic response by monocytes, of the
NBT-ET Stress 14.8 3.11 0.51
phagocytosis digestion phase by neutrophils, of the
Control 14.2 2.65
IgA, IgM, IgG, and C3 and C4 complement compo-
IgA Stress 272.7 134.22 0.54 nent serum levels during acute stress.
Control 299.43 153.08
IgM Stress 154.9 75.33 0.71
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