Changes in the activated cytotoxic suppressor T-lymphocytes in elderly patients with mediterranean spotted fever.

INTRODUCTION

The traditional concept of Mediterranean spotted fever (MSF) is that it is a benign disease. Nowadays, several peculiarities in the spread and the clinical course of MSF are observed worldwide, with increase of the severe cases and complications. (1) MSF has been the most common tick-borne rickettsiosis in Bulgaria for the last 10 years. MSF emerged in Varna in 1994. Since then more than 1700 people have been affected and more than 993 of them have been hospitalized in the Department of Infectious Diseases, University Hospital, Varna, Bulgaria. Cases of moderate severity predominated (53,10% - 470 patients), followed by the severe forms (20,90% - 185 patients), 16 of which died--1,61%. (2) The severity of the disease and the complications reached highest expression in patients of over 60, according to our experience, who represent the 30,21% (300 patients). (34)

Clinical observations of the ageing process lead to the conclusion that old age is the most widely distributed type of immunodeficiency. The ageing process affects the immune system at all levels: chemical changes in cellular biological structures, differences in the types of the surface cell proteins, changes in the organs. (5,6)

Ageing affects the function of T-cells with multiple biological processes. Spotted fever group rickettsiae stimulate both cell mediated and Immoral immunity, but T/CD8+/ lymphocytes are the primary effector cells of the immune response and the activity of the cytotoxic suppressor T-lymphocytes play a primary role for the healing process. (7)

In this study we investigated the cell immunity of 20 patients over 60 with Rickettsiae conorii infection. The aim of this research was to specify the changes in the cytotoxic suppressor T- lymphocytes in elderly patients with MSF.

METHODS

A total of 132 patients over 60 years (60+) during 2000-2007 with MSF were enrolled in the study. Thirty patients under 60 years of age (20-54 years old) served as a control group. The diagnosis of MSF was established following the Guidelines of ESCAR (ESCMID Study Group on Coxiella, Anaplasma, Rickettsia and Bartonella) and the European Network for Surveillance of Tick-borne Diseases.

The severe forms of the disease were characterized with symptoms presented in Table 1, plus several neurologic manifestations, heart failure, hemorrhagic rash with significant reduction in the platelets count and fibrinogen levels, as well as markers of acute renal failure. The severity of the clinical picture was even worsened by the development of multiorgan failure.

Hematological and biochemical investigations were performed in the laboratory of the Department of Infectious Diseases. The diagnosis was serologically confirmed using immunofluorescent assay.

We investigated the cell immunity of 20 patients of age over 60 years in the same cohort, using immunofluocytometry. The control group consisted of 10 young patients with MSF and 10 healthy individuals over the age of 60. Venous blood samples were collected with the use of closed system with vacutainer of 5m1 and anticoagulant Na-heparin. Monoclonal antibodies, conjugated with FITC (Fluoresceini-sothiocyanat) or PE (Phycoerythrin), served for immunophenotyping from pannel-kit IMK--Plus (immune monitoring kit -Becton-Dickinson), which is recommended by the WHO and includes: LeucoGATE CD45 FITC/CD45 PE, isotype control IgG1 FITC/IgG2PE, and for expression of the surface lymphocytes antigens:

1. Main lymphocyte populations: Common T-lymphocytes (CD3+), Common B-lymphocytes (CD19 +), Natural killers (CD16+56+).

2. Lymphocyte subpopulations: Th - Helper/Inducer (CD4+), Ts - Suppressor / Ctotoxic (CD8+).

3. Activation of T-lymphocytes:

* Late activation of the common T-lymphocytes (CD3+/HLA-DR+)

* Late activation of Ts Suppressor / Ctotoxic T- lymphocytes: (CD8+/CD38+)

In this study we interpret the changes in CD8+/CD38+ Ctotoxic T- lymphocytes.

Lymphocyte processing

Standard procedure for direct two-colour immunofluorescence with whole lysed blood: washing with CellWash, lysis with Lysis Solution and fixing with 1,5% formaldehid- CellFix (BD). The examination was performed on floucytometer FACSort (Becton-Dickinson-USA) with the capacity to read simultaneously five parameters. The results were automatically analyzed using SimulSet softwear product (Verity Softwear House, Inc.). Minimum 50000 cells were analyzed. Variation, alternative, nonparametric, correlative and graphic analysis was used during the statistical processing.

RESULTS--DISCUSSION

Clinical characteristics

According to our own observation of the hospitalized patients (2-3) and using adapted criteria, already established by other authors, 3 clinical groups with different severity of the course were formed. (7,8,9,10) The major clinical symptoms of all observed patients are shown in Table 1.

Decreased adaptive abilities, changed homeostatic mechanisms make people of 60+ extremely susceptible to the infection with R.conorii. In 79,54% (105) of the patients of 60+ different underlying conditions were present: cardiovascular disease, cerebrovascular disease, chronic obstructive pulmonary disease, pneumopathia, diabetes mellitus, renal disease. The 43,18% (57) of the elderly patients presented with severe form, the 46,21% (61)--with moderate form, 10,61% (14)--with mild form of the disease (see figure 1).

Thirteen of the 132 studied elderly patients had mental changes and 3 were in coma during the first days. Three patients suffered from focal neurological symptoms (pareses and seizures) and 4 patients over 60 presented with signs of meningeal irritation. The above conditions were estimated as a result of the development of cerebral edema with meningismus, and as a result of stroke in one case. Thirty one patients (23,48%) of the group over sixty had disturbances in the renal function -11 patients in a mild form and 20 in a severe form. In the control group only 2 patients were diagnosed to have mental changes, 3 presented with paresis and 3 with meningismus in the first days. Only three of the younger ones (10%) had disturbances of the renal function in mild form. MSF had a fatal outcome in 13 (1,30%) of the hospitalized elderly patients. The presence of chronic diseases pre-determine the clinical course and outcome of MSF in persons over 60 years of age.

In the immunologically investigated group of 20 patients 60+ during 2006-2007, similar clinical conditions have been observed. With severe form of MSF were presented 5 persons, with moderate form 12 and with benign illness 3 (see Table I a).

Immunological features

Significantly more increased expression of CD8+/CD38+ was found in the elderly patients (22,35+/-4,71), compared to the patients of the control group (22,35+/-5,76) for the young patients and (22,43+/-7,31) for the healthy persons over 60 years (p<0,05 for both).The observed differences in the CD8+CD38+ cells expression are statistically reliable (see Table 2).

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CD8+CD38+ in the three groups under investigation are in straight correlative dependence of the age (See figures 2, 3, 4).

The dependence is significant in healthy elderly persons of the control group (r=0,59), moderate in young patients (r=0,37) and low in patients 60+ (r=0,03), because in the case of the last mentioned, other factors exert influence too, for example the severity of the clinical course.

In elderly patients with MST, CD8+CD38+ are in moderate (r=0,34) reverse correlative dependence on the severity of the disease (see figure 5), whereas in the young patients investigated, activated cytotoxic suppressor T-lymphocytes are in strong /r=0,87/ straight dependence on the severity of the disease (see figure 6).

The primary role of the activated CD8+T-lymphocytes is clearly documented in experiments with MHC-class I--knockout mice. (11) The quantity of the apoptosal cells in the liver of the infected animals is significantly higher in those which received immune CD8+T-lymphocytes, than in the control group of animals which received non-immune CD8+T- lymphocytes. This probably is a result of cytotoxic T- lymphocyte mediated apoptosis of the infected cells. (12) Other cytotoxic T- lymphocyte mechanisms are regarded to play a role as well, such as Fas/Fas ligands or granulysis (a protein, discovered in granules of cytotoxic T-lymphocytes, which in combination with perforin reduces the vitality of a broad spectrum of pathogenic bacteria and through which T- cells directly contribute to the immunity against intracellular pathogens). (7) Also, it is possible that the immune CD8+T-lymphocytes exercise immunopathological effect in cases of very extensive infection of the endothelial cells in the time of the lymphocyte clonal expansion and activation." CD8+T- cells could play double role (immune clearance and immune pathology), depending on the antigenic load. Our investigations also confirm the significance of the activity of cytotoxic T-lymphocytes for the healing process in elderly persons with MSF.

CONCLUSIONS

1. Expression of the activated cytotoxic suppressor T-lymphocytes in elderly patients with MSF is increased.

2. Expression of CD8+CD38+ correlates with the severity of the disease.

REFERENCES

(1.) Amaro M, Bacellar F, Franca A. Report of eight cases of fatal and severe Mediterranean spotted fever in Portugal. Ann N Y Acad Sci. 2003;990:331-43.

(2.) Annual reference book. Regional Inspection for Control of Environment and Public Health. Varna: National Center of Public Health Protection, 2006.

(3.) Gospodinova M, Nenova M. Mediterranean spotted fever in adults aged over 60 years. Scripta Scientifica Medica 2003;35:91-94.

(4.) Nenova M. Mediterranean Spotted Fever in Varna Region, in Contemporary State of the Rickettsioses in the World and in Bulgaria. Sofia: 2007, pp 94-106.

(5.) Whitman DB (1999). The immunology of aging. Cambridge Scientific Abstracts. http://www.csa.com/hottopics/immune-aging/overview.html. Last accessed at January 28, 2002.

(6.) Meyer O. Immunologic et vieillissement [Immunology of ageing] Geriatrics praticiens et 3eme age 1994;10:162-167 [Article in French].

(7.) Raoult D, Weiller PL Chagnon A, Chaudet H, Gallais H, Casanova P. Mediterranean spotted fever: clinical, laboratory and epidemiological features of 199 cases. Am J Trop Med Hyg 1986;35(4):845-850.

(8.) Popivanova N, Baltadziev I, Zaprianov Z. Mediterranean spotted fever in the Plovdiv region of Bulgaria. In: Contemporery state of the rickettsioses in the world and in Bulgaria. Sofia: Publication of the Bulgarian Academy of Sciences, 2007: pp116-124.

(9.) Raoult D, Kohler J L, Gallais H, De Micco P, Rousseau S, Casanova P. [Fatal rickettsioses]La Nouvelle presse medicale 1982;11(8):607.

(10.) Raoult D, Zuchelli P, Weiller PL Chattel C, San Marco JL, Gallais H, Casanova P. Incidence, clinical observations and risk factors in the severe form of Mediterranean spotted fever among patients admitted to hospital in Marseilles 1983-1984. J Infect. 1986;12(2):111-116.

(11.) Valbuena, G., H.-M. Feng, and D. H. Walker. Mechanisms of immunity against rickettsiae. New perspectives and opportunities offered by unusual intracellular parasites. Microbes Infect. 2002;4:625-633.

(12.) Valbuena G, Walker DH. The endothelium as a target for infections. Annu Rev Pathol. 2006;1:71-98.

(13.) Valbuena G, Bradford W, Walker DH. Expression analysis of the T-cell-targeting chemokines CXCL9 and CXCL10 in mice and humans with endothelial infections caused by rickettsiae of the spotted fever group. Am J Pathol. 2003;163(4):1357-69.

Margarita D Gospodinova, Marina A Nenova, Sevda S Mileva

Medical University of Varna, Bulgaria

Corresponding author: Margarita D Gospodinova, Address: 61 Dr Basanovich Str, Floor 1, ap.97 Var a 9010, Bulgaria e-mail: [e-mail]

Table 1. Major symptoms in patients over 60 years of age with MSF.

Symptoms                                 Patients     Control
                                         60+n=132    group n=30
                                                      (20-54)
                                                     years old

Fever:
Up to 38 [degrees]                        20,70%       50,00%
Over 39 [degrees]                         79,20%       50,00%
Up to the third day                       32,40%       40,00%
Up to the fifth day                       32,40%       40,00%
Up to the seventh day                     22,00%       10,00%
Up to the 10-th day and more              12,90%       10,00%
Tache noire                               88,30%       63,30%
Conjunctival and pharyngeal injection     84,40%       73,30%
Rash:
Papulous                                  3,80%        6,66%
Maculopapular                             77,27%       80,00%
Hemorrhagic                               18,93        13,34
Abundant                                  88,30%       63,30%
scanty                                    11,60%       36,60%
Leucocytosis                              15,50%       20,00%
Low platelets count                       36,30%       23,30%

Symptoms                                    t            p

Fever:
Up to 38 [degrees]                         2,8         < 0,05
Over 39 [degrees]                          2,8         < 0,05
Up to the third day                        0,73        > 0,05
Up to the fifth day                        0,73        > 0,05
Up to the seventh day                      1,66        > 0,05
Up to the 10-th day and more               0,44        > 0,05
Tache noire                                2,63        < 0,05
Conjunctival and pharyngeal injection      1,2         > 0,05
Rash:
Papulous                                   0,47        > 0,05
Maculopapular                              0,57        > 0,05
Hemorrhagic                                0,64         0,05
Abundant                                   2,63        < 0,05
scanty                                     1,94        < 0,05
Leucocytosis                               0,54        > 0,05
Low platelets count                        1,38        > 0,05

Table 1a. Major clinical symptoms in the immunologically investigated
patients 60+ with MSF.

Symptoms                                Mild clinical       Moderate
                                        form, n=3(15%)   clinical form,
                                                           n=12(60%)

Fever:
T--up o t 38[degrees] C                     3-100%             --
T--38[degrees]-38,5[degrees] C                --            7-58,33%
T--over 39[degrees] C                         --            5-41,67%
Tache noire                                 3-100%          12-100%
Conjunctival and pharyngeal injection      2-66,67%        10-83,33%
Papulous                                      --            1-8,33%
Maculopapular                               3-100%           9-75%
Purpura                                       --            2-16,67%
Abundant rash                                 --            12-100%
Scanty rash                                 3-100%             --
Leucocytosis                                  --             3-25%
Leucopenia                                    --            1-8,33%
Left shift                                 2-66,67%         7-58,33%
Low platelets count                           --            5-41,66%
Fibrinogen level under 2g/l                   --            2-16,67%

Symptoms                                    Severe        Investigated
                                        clinical form,   patients, n=20
                                           n=5(25%)

Fever:                                                      20-100%
T--up o t 38[degrees] C                       --             3- 15%
T--38[degrees]-38,5[degrees] C                --             7-35%
T--over 39[degrees] C                       5-100%           10-50%
Tache noire                                 5-100%          20-100%
Conjunctival and pharyngeal injection       5-100%          17- 85%
Papulous                                      --             1- 5%
Maculopapular                               3-60%           15- 75%
Purpura                                     2-40%            4- 20%
Abundant rash                               5-100%           17-85%
Scanty rash                                   --             3-15%
Leucocytosis                                2-40%            5-25%
Leucopenia                                  1-20%            2-10%
Left shift                                  4-80%            13-65%
Low platelets count                         3-60%            8-40%
Fibrinogen level under 2g/l                 1-20%            3-15%

Table 2.1. Comparative analysis of the immune indexes in patients
with MSF--60+ and younger patients

Immune      Patients with MSF over 60
indexes     (x [+ or -] [sigma]) n=20

CD3+        69,63    [+ or -] 11,67
CD19+       11,43    [+ or -] 6,11
CD4+        44,05    [+ or -] 10,29
CD8+        41,19    [+ or -] 9,48
AKT. T.     27,45    [+ or -] 10,86
NK          20,16    [+ or -] 9,45
CD38+       72,47    [+ or -] 13,34
CD8+CD38+   31,04    [+ or -] 10,08
CD4+/CD8+    1,22    [+ or -] 0,43

Immune      Young patients with MSF
indexes     (x [+ or -] [sigma]) n=10

CD3+        66,42    [+ or -] 10,42
CD19+       12,61    [+ or -] 3,57
CD4+        45,62    [+ or -] 10,96
CD8+        35,76    [+ or -] 9,12
AKT. T.     21,35    [+ or -] 9,12
NK          17,02    [+ or -] 4,21
CD38+       72,91    [+ or -] 11,53
CD8+CD38+   22,35    [+ or -] 8,05
CD4+/CD8+    1,59    [+ or -] 0,59

Immune        t         p
indexes

CD3+         0,76    > 0,05
CD19+        0,66    > 0,05
CD4+         0,37    > 0,05
CD8+         1,52    > 0,05
AKT. T.      1,62    > 0,05
NK           1,26    > 0,05
CD38+        0,09    > 0,05
CD8+CD38+    2,55    < 0,05
CD4+/CD8+    1,95    < 0,05

Table 2.2. Comparative analysis of the immune indexes in patients
with MSF--60+ and healthy persons 60+

Immune      Patients with MSF over 60
indexes     (x [+ or -] [sigma]) n=20

CD3+        69,63    [+ or -] 11,67
CD19+       11,43    [+ or -] 6,11
CD4+        44,05    [+ or -] 10,29
CD8+        41,19    [+ or -] 9,48
AKT. T.     27,45    [+ or -] 10,86
NK          20,16    [+ or -] 9,45
CD38+       72,47    [+ or -] 13,34
CD8+CD38+   31,04    [+ or -] 10,08
CD4+/CD8+    1,22    [+ or -] 0,43

Immune       Healthy persons over 60
indexes     (x [+ or -] [sigma]) n=10

CD3+        72,55    [+ or -] 8,10
CD19+        7,57    [+ or -] 2,67
CD4+        46,83    [+ or -] 10,29
CD8+        39,67    [+ or -] 8,30
AKT. T.     11,35    [+ or -] 2,80
NK          21,49    [+ or -] 7,16
CD38+       64,21    [+ or -] 10,61
CD8+CD38+   22,43    [+ or -] 10,22
CD4+/CD8+    1,36    [+ or -] 0,52

Immune        t      p
indexes

CD3+         0,80    > 0,05
CD19+        2,41    < 0,05
CD4+         0,69    > 0,05
CD8+         0,45    > 0,05
AKT. T.      6,24    < 0,001
NK           0,14    > 0,05
CD38+        1,84    > 0,05
CD8+CD38+    2,18    < 0,05
CD4+/CD8+    0,74    > 0,05

Table 2.3. Comparative analysis of the immune indexes in young
patients with MSF healthy persons over 60 years of age

Immune      Young patients with MSF
indexes     (x [+ or -] s) n=10

CD3+        66,42    [+ or -] 10,42
CD19+       12,61    [+ or -] 3,57
CD4+        45,62    [+ or -] 10,96
CD8+        35,76    [+ or -] 9,12
AKT. T.     21,35    [+ or -] 9,12
NK          17,02    [+ or -] 4,21
CD38+       72,91    [+ or -] 11,53
CD8+CD38+   22,35    [+ or -] 8,05
CD4+/CD8+    1,59    [+ or -] 0,59

Immune      Healthy persons over 60
indexes     (x [+ or -] [sigma]) n=10

CD3+        72,55    [+ or -] 8,10
CD19+        7,57    [+ or -] 2,67
CD4+        46,83    [+ or -] 10,29
CD8+        39,67    [+ or -] 8,30
AKT. T.     11,35    [+ or -] 2,80
NK          21,49    [+ or -] 7,16
CD38+       64,21    [+ or -] 10,61
CD8+CD38+   22,43    [+ or -] 10,22
CD4+/CD8+    1,36    [+ or -] 0,52

Immune        t      p
indexes

CD3+         1,47    > 0,05
CD19+        3,57    < 0,05
CD4+         0,25    > 0,05
CD8+         1,0     > 0,05
AKT. T.      3,31    < 0,05
NK           1,69    > 0,05
CD38+        1,76    < 0,05
CD8+CD38+    0,02    > 0,05
CD4+/CD8+    0,92    > 0,05

* [sigma]--median standard deviation

Figure 1. Clinical forms according to the symptom severity in %.

         Severe form   Moderate form   Mild form

Ha 60+      43,18           46,21        10,61
< 60           20              50           30

Note: Table made from bar graph.