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Lymphocyte Population in Patients With Tularemia
J. L. Pérez-Castrillón, MD*
M. Martín-Luquero, MD*
P. Bachiller, MD*
J. Mena, MD*
I. Recio, MD*
A. Jimenez, MD†
M. Romero, MD†
V. Herreros, MD
*Department of lnternal Medicine, Río Hortega University Hospital, University of Valladolid, Valladolid, Spain
†Department of Hematology, Río Hortega University Hospital, University of Valladolid, Valladolid, Spain
KEY WORDS: tularemia, lymphocyte population, prognosis
Background: The objective of study was to evaluate the evolution of lymphocyte population in patients with tularemia before initiating treatment and 1 year later and to evaluate their prognostic value.
Patients and Methods: The patients were diagnosed of tularemia by compatible clinical manifestations and by an elevated titer of antibodies against Francisella tularensis (>1/160), seroconversion or isolation of F. tularensis in a clinical sample. The control group consisted of asymptomatic patients with chronic ischemic heart disease with negative serology against F. tularensis and Chlamydia pneumoniae. The patients were treated with streptomycin or ciprofloxacin. The following cell populations were determined by FACSCAN cytometer: T lymphocytes, helper T lymphocytes, suppressor T lymphocytes, activated T lymphocytes, T lymphocytes with NK activity, B lymphocytes, and NK cells. The progression was evaluated using clinical criteria.
Results: During the initial determination, the patients presented statistically significant elevated values of total lymphocytes, T lymphocytes, activated T lymphocytes, and T lymphocytes with NK activity. The values had become normal 1 year after termination of treatment. There were no differences between those patients with a poor response to treatment and those with a positive one.
Conclusions: Patients with tularemia initially presented a high total lymphocyte count, T lymphocytes, activated lymphocytes, and T lymphocytes with natural killer activity. The cell populations lack value as a prognostic marker.
Tularemia is a zoonosis caused by Francisella tularensis, a gram-negative coccobacillus, carried by lagomorphs and small rodents, which can infect domestic animals, birds, rodents, and people. The presentation of the disease can either be sporadic or epidemic.1 People can enter into contact with the microorganism in various ways: contact, ingestion, or inhalation of aerosols, the clinical presentation depending on the portal of entry. Once the bacteria has penetrated the organism, the humoral and cellular response mechanisms are activated. Initially, there is an important increase in serum antibodies in response to the microorganism, but this increase does not serve to protect the host, although it is useful to diagnose the disease. Effective protection is achieved by a cellular mechanism that can be divided into 2 phases: an initial T lymphocyte-independent phase and another later T-dependent phase.2
The objective of this study is to evaluate the progressive profile of the lymphocyte subpopulation in patients with tularemia during the first months after infection and 1 year after having completed treatment. We also want to determine the prognostic value of the lymphocytic pattern to attempt to identify those patients with poor progress and to be more therapeutically aggressive with this group.
Patients and Methods
Twenty-nine patients affected by an epidemic outbreak of tularemia in the region of Castilla y León, Spain, during November and December of 1997 were studied.3 The patients were diagnosed with tularemia as a result of compatible clinical symptoms and one of the following laboratory criteria: antibody titer against F. tularensis antigen >1/160 in one or more samples from the onset of symptoms, seroconversion, and isolation of F. tularensis from a clinical sample. Serologic testing was done by means of the standard tube agglutination test and the culture was made in a media of cysteine-enriched blood agar. The age, sex, clinical manifestations (ulceroglandular, glandular, typhoidal, oropharyngeal, oculoglandular, and pulmonary), time from the onset of clinical symptoms until the patient was examined in our hospital clinic, and response to treatment for each patient was evaluated. The response was judged good or poor. The response was classified as poor under the following conditions: continuing or reappearance of fever, persistence, increase in size or appearance of new adenopathies, and persistence of symptoms with an increase in acute-phase reactants. The patients were studied before treatment was initiated and again 1 year after antibiotic treatment was finished. Treatment consisted of streptomycin (500 mg/12 hours intramuscularly for 10 days) or ciprofloxacin (750 mg/ 12 hours given orally for 2 weeks). Whole blood with edetic acid was obtained from each patient. The following MoAbs were used in immunofluorescence studies: CD3+/CD5/CD2+ (T lymphocytes), CD4+ (T helper lymphocytes), CD8+ (T-suppressor lymphocytes), CD3+/HLA-DR+ (activated T lymphocytes), CD3+/CD8+CD56+ (T lymphocytes with NK activity), CD 19+/HLA-DR (B lymphocytes), and CD16+/CD56+/CD2+ (NK cells). All antibodies were obtained from Becton-Dickinson (San Jose, CA). Phycoerythrin (PE) -, fluorescein isothiocyanate (FITC) -, or PerCP-conjugated MoAbs were used for fluorescence staining of whole blood samples. Cells were analyzed on a FACSCAN flow cytometer (Becton-Dickinson). As a control group, cell population was determined in 17 asymptomatic patients of similar age with chronic ischemic heart disease, stable angor, and with negative antibodies against F. tularensis and Chlamydia pneumoniae.
The data were processed on a PC Pentium 166 Mz with an SPSS 7.0 program (Chicago, IL) using the Wilcoxon test as a statistical test for paired data and the U of Mann-Whitney for nonpaired data.
The characteristics of the patients studied are described in Table 1. In Table 2, it can be observed that in the first determination the patients presented statistically significant high levels of total lymphocytes, T lymphocytes, activated T lymphocytes, and T lymphocytes with NK activity. The cell population determined 1 year after finishing antibiotic treatment presented no differences with respect to the control group. There were no variations in cell population in relation to age, sex, clinical manifestation of the disease, evolution time, or response to treatment.
In the initial extraction, the cell population presented an increase in total lymphocyte count, T lymphocytes, activated T lymphocytes, and T lymphocytes with NK activity. In the determination made 1 year after cessation of treatment, these disorders had disappeared, and no differences relative to the control group were observed. The majority of the immunologic studies of F. tularensis have been carried out with murine subjects, because rodents constitute the principle reservoir of the organism.4 Fewer studies have been undertaken in humans with tularemia. During a bibliographic search, no reference to the use of a lymphocyte population for the prognostic evaluation of the disease was found.
Francisella tularensis is an intercellular microorganism that triggers a humoral and cellular response in the host. During the initial phase, the type of cell that intervenes is not precisely known, and although the neutrophils can play an important role, because they release TNFa that attracts macrophages and T lymphocytes to the infected area, those cells most frequently implicated are the NK cells.5,6 In in vitro studies of mononuclear populations of tularemia patients, the only cytokine produced during the first week was TNFa.7 Our results show NK circulating values within normal limits in the 2 determinations undertaken and without differences with respect to the control group. A possible explanation is the sequestration of these cells in the lymphatic ganglia, because 80% of the patients had ganglionar involvement. Nevertheless, a more probable hypothesis is that of the time elapsed from the onset of symptoms to the extraction, an average of 10 weeks. When the determination has been made, other T-dependent mechanisms have already been set in motion. The blastic transformation and the secretion of cytokines is initiated during the second phase of the disease that begins between the first and fourth weeks and is mediated by stimulated T lymphocytes.8,9 The population that measured activated T lymphocytes (CD3-HLA DR+) showed a statistically significant increase in the first determination studied, the macrophage phagocyte F. tularensis, processing it and presenting on the surface antigen determinants associated with the major class II histocompatibility gene complex (MHC II). The T lymphocytes that are activated are CD4, which transform into Th1 and produce IL-2 and interferon g.7 The former produces a clonal expansion of the cytotoxic CD8 cells and the interferon g activates the infected macrophages to stop growth of the microorganism through the generation of toxic levels of nitric oxide and to facilitate their destruction.10,11 Our patients had high levels of cytolytic T Iymphocytes or T lymphocytes with NK activity, cells that recognize and destroy the antigen-presenting cell that induced their activation.
The number of CD4 and CD8 underwent no changes, despite the fact that the response to the specific antigen is CD4-dependent, selectively of TH1 cells. Our study is quantitative and not qualitative. The expansion of gdT cells has not been studied, cells that do not express CD4 or CD8 molecules. Sumida et al. demonstrated an expansion of this population in a patient with tularemia.12 The role they could play in this infection is unknown, but it is possible that they collaborate with the CD4 in cellular defense mechanisms. The mechanisms described are maintained for many years, and the lymphocytes of patients infected with F. tularensis retain the capacity to respond to membrane proteins of the microorganism and to induce the production of TH1-dependent cytokines.13
A second aspect that we wanted to evaluate was whether the virulence of the germ held any relation to an inadequate development of the cellular protection mechanisms. In the group of patients studied, there was a high percentage (37%) with a poor response to antibiotic treatment. Nevertheless, this group did not present a cell population pattern different from the rest. This data is compatible with that observed in a murine model by Golovliov et al., who demonstrated that the mortality rate of the infection did not depend on the host response, but rather on the virulence of the microorganism.4 In our population, the high percentage of patients with a poor response was probably related to the diagnostic delay (10 weeks). The number and balance of lymphocyte population, which lack use as a prognostic indicator of the disease, was not a determining factor.
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4. Golovlov I, Kuoppa K, Sjösted A, Tärnvik A, Sandström G: Cytokine expression in the liver of mice infected with a highly virulent strain of Francisella tularensis. FEMS Immunol Med Microbiol 13:239–244, 1996.
5. Tärnvik A, Ericsson M, Golovliov I, Sandström G, Sjösted A: Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis as a model organism. FEMS Immunol Med Microbiol 13:221–225, 1996.
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8. Tärnvik A, Eriksson M, Sandström G, Sjösted A: Francisella tularensis—a model for studies of the immune response to intracellular bacteria in man. Immunology 76:349–354, 1992.
9. Tärnvik A, Sandström G, Lofgren S: Time of lymphocyte response after onset of tularemia and after tularemia vaccination. J Clin Microbiol 10:845–860, 1979.
10. Fortier AH, Polsinelli T, Green SJ, Nacy CA: Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules. Infect Immun 60:817–825, 1992.
11. Conlan W, Sjösted A, North RJ: CD4+ and CD8+ T-cell-dependent and independent host defense mechanisms can operate to control and resolve primary and secondary Francisella tularensis LVS infection in mice. Infect Immun 62:5603–5607, 1994.
12. Sumida T, Maeda T, Takahashi H, et al: Predominant expansion of Vy9/ V52 T cells in a tularemia patient. Infect Immun 60:2554–2558, 1992.
13. Ericsson M, Sandström G, Sjösted A, Tärnvik A: Persistence of cell-mediated immunity and decline of humoral immunity to the intracellular bacterium Francisella tularensis 25 years after natural infection. J lnfect Dis 170:110–114, 1994.
Table 1. Characteristics of the Population Under Study
Age 52 ± 15
Evolution time (mo) 2.15 ± 1.3
Response to treatment
Table 2. Lymphocyte Population in Patients With Tularemia
Initial Final Control
Lymphocytes 2786 ± 93* 2182 ± 624 2183 ± 687
CD3/CD5/CD2 2187 ± 798* 1612 ± 528 1498 ± 512
CD4 962 ± 385 870 ± 342 823 ± 260
CD8 533 ± 297 398 ± 178 562 ± 391
CD3/MHC-DR 472 ± 332 53 ± 41 66 ± 42
CD3/CD8/CD56 240 ± 224 152 ± 209 91 ± 139
CD19/MHC-DR 186 ± 111 213 ± 85
294 ± 143 216 ± 162 401 ± 198
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