Th1/Th2 Milieu in Adult Hemophagocytic Syndrome

Abstract

Clinically the hemophagocytic syndrome (HPS) is characterized by high fever, hepatosplenomegaly, cytopenia and coagulopathy, and histologically by increased proliferation and activation of histiocytes/macrophages [1, 2, 3]. Although the clinical entity of this syndrome has been established, its pathogenesis is still poorly understood.Many reports, however, suggest that abnormalities in the immune regulatory system play a central role in HPS [2, 3]. The elevated levels of soluble (s) interleukin-2 receptor (sIL-2R) and sCD8 in active HPS and the normal levels observed in remission indicate that an activation of T cells, especially CD8+ T cells (cytotoxic T cell, Tc cells) contribute to the development of HPS [4]. Serum concentrations of various cytokines [IL-1, IL-2, IL-6, tumor necrosis factor-α (TNF-α), interferon (IFN)-α, IFN-γ, macrophage colony-stimulating factor, IL-1 receptor antagonist] are often elevated [reviewed in ref. 1, 2, 3]. In particular, IFN-γ is one of the major cytokines elevated in HPS, and its level reflects the activity of the disease [5].Distinct cytokine profiles are clearly associated with and related to clinical processes of certain infectious and immunological diseases. Cytokine profiles are determined to some extent by two functional subsets of T helper cells designated Th1 and Th2 [6, 7, 8]. Th1 cells secrete IL-2, TNF-β and IFN-γ and stimulate production of antibodies of the IgG2a class, which mainly trigger phagocyte-mediated host defense against intracellular pathogens. On the other hand, the Th2 subset, which produces IL-4, IL-5, IL-10 and IL-13, is mainly responsible for phagocyte-independent host defense against extracellular pathogens. Immune responses to pathogens often generate polarization of T helper subsets, which leads to protection against pathogens or progression of inflammatory processes.Immunological findings observed in HPS suggest that an imbalance in Th1-Th2 cells, especially uncontrolled augmentation of Th1 response and subsequent activation of Tc cells and macrophages, may be the culprit in its pathophysiology [1, 2]. A recent report supported polarization toward a Th1 response in HPS by showing that the serum level of IL-12, which induces Th1 response, is elevated, while that of IL-4, which promotes Th2 response, is not [9]. To further elucidate findings of an imbalance in Th response, Th subsets in peripheral blood from patients with HPS were evaluated.Patients subjected to the present study include 7 virus-associated HPS (VAHS, group A), 7 virus infections without HPS (group B) and 5 bacteria-associated HPS (BAHS, group C; table 1). Diagnosis of HPS was based on our own criteria [1, 2]. Measurement of Th subsets was performed by flow-cytometric determination of cytoplasmic IFN-γ and IL-4 in peripheral CD4-positive cells. Briefly, whole blood was incubated with phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A (Sigma, St. Louis, Mo., USA) for 4 h according to the method by Picker et al. [10]. Then, cells were stained with peridinin chlorophyll protein-labelled anti-CD4 (Becton Dickinson Immunocytometry Systems, BDIS, San Jose, Calif., USA). After treatment with FACS lysing solution and FACS permeabilizing solution (BDIS), cells were stained with FITC-labelled anti-human IFN-γ and PE-labelled anti-human IL-4 (BDIS) [11]. FITC-mouse IgG2a and PE-mouse IgG1 were used as controls. Percent of IFN-γ- and IL-4-positive cells were counted by FACScan (BDIS) and the Th1/Th2 balance was evaluated with a ratio of percent IFN-γ single-positive cells (Th1) to percent IL-4 single-positive cells (Th2). Referential values of normal healthy controls (mean ± SE, n = 20, 24–52 years, average 37 years) were as follows: Th1 19.91 ± 1.21%, Th2 2.74 ± 0.24%, and Th1/Th2 8.04 ± 0.71 (table 2).The percentages of lymphocytes with CD4 and CD8 markers are shown in table 1 (normal ranges: 35–53 and 19–32%, respectively). In viral infections (groups A and B), there were tendencies for CD4+ cells to decrease (33.9 ± 3.1 and 26.7 ± 6.3%, respectively), for CD8+ cells to increase (42.1 ± 4.0 and 43.2 ± 8.6%), and consequently for a reduced CD4/CD8 ratio (0.86 ± 0.13 and 0.95 ± 0.06). However, there were no differences in these parameters between groups A and B (p > 0.05, unpaired Student’s t test). Therefore, it is suggested that the changes in the CD4/CD8 subsets induced by viral infections were not influenced by the presence of HPS. In BAHS (group C), all these parameters, were within normal ranges: CD4+ cells 50.2 ± 9.2%, CD8+ cells 26.1 ± 6.7% and a CD4/CD8 ratio of 2.66 ± 1.0. Thus, there was not a definite tendency for CD4/CD8 subsets to fluctuate in either VAHS or BAHS.Table 2 shows the Th subsets in patients in each group. There were no differences in either percent Th0, Th1 or Th2 or in Th1/Th2 ratios between groups A and B: Th0, 1.84 ± 0.41 vs. 1.42 ± 0.29%; Th1, 21.20 ± 2.70 vs. 17.40 ± 3.02%; Th2, 2.91 ± 0.46 vs. 3.06 ± 0.66%, and Th1/Th2 ratio, 7.22 ± 1.18 vs. 8.16 ± 2.28 (p > 0.05 in all combinations). In addition, neither percent Th nor Th1/Th2 ratios in groups A and B differed from those of healthy controls (p > 0.05). On the other hand, samples from BAHS (group C) showed a lower concentration of Th1 (12.51 ± 2.19%), a higher concentration of Th2 (4.08 ± 0.56%) and consequently a much lower Th1/Th2 ratio (3.08 ± 0.26) than those from VAHS (group A) and normal controls. The differences in Th1 concentrations and Th1/Th2 ratios between groups A and C were significant (p < 0.05 and p < 0.02, respectively). When group C and normal controls were compared, differences in Th1 and Th2 concentrations and in the Th1/Th2 ratio were all statistically significant (p < 0.02, p < 0.05, and p < 0.01, respectively).These results indicate, contrary to our expectation, that polarization of the Th subset to Th1 is at least not a general observation in HPS as far as peripheral lymphocytes are concerned. In VAHS, the levels of Th0, Th1, Th2 and Th1/Th2 were comparable to those in viral infections without HPS and even in healthy controls. Furthermore, Th1/Th2 profiles did not reflect the clinical activity of the disease in case 2 in whom samples in active and remission phases could be examined (tables 1, 2, data from the remission phase are in parentheses). Therefore, it is not plausible that the Th1 shift is causally involved in VAHS at least at present. Reportedly, acute viral infections show various Th1/Th2 profiles probably depending on the type of infecting virus [6, 7, 8]. However, even when the same Epstein-Barr virus infections were compared, the differences in Th1/Th2 profiles between group A (cases 1–3) and group B (cases 8–10) were not remarkable (table 2).In BAHS, conversely, a significant decrease in the Th1/Th2 ratio was observed, which implies a shift to Th2. This reduction in the Th1/Th2 ratio resulted from both decrease in Th1 and increase in Th2 concentrations (table 2). Human bacterial diseases, other than leprosy and tuberculosis, have not yet been intensively analyzed in terms of Th1/Th2 profiles [12]. Therefore, further study is required to explain this Th2 shift in BAHS. In any case, this marked difference in Th1/Th2 profiles between VAHS and BAHS suggests a difference in the immunological milieu between these two categories of HPS. When immunosuppressive agents are used to treat HPS, attention should be paid to this difference in the immunological aspects in each type of HPS [2].Recently, infectious disease systems and other immunological phenomena have been elucidated with increasing sophistication. The Th1-Th2 paradigm is sometimes recognized as an oversimplification [13]. One of the reasons is that several Th1 and Th2 cytokines have been shown to originate not only from CD4+ T cells but also from numerous other leukocytes and even from non-hematopoietic cells [6, 8, 13]. Therefore, type 1 or Th1-like cytokine dysregulation in HPS does not always indicate a CD4+ Th1 response or an increased Th1/Th2 ratio. In addition, Th1/Th2 profiles in peripheral blood may not necessarily reflect a possible localized or compartmentalized dysregulation of type 1/type 2 cytokines. It might be important to avoid immediate categorization of HPS into Th1-type or Th2-type responses; instead immunological responses by individual cytokines and the effector pathway should be assessed.Flow-cytometric analyses were performed by Drs. Tsutomu Kohsaka, Yuji Kudoh, Toshiyasu Miyazaki and Masahiro Oyamada, Otsuka Tokyo Assay Laboratories.