Mechanisms of Interleukin-2-Mediated Signaling and role of Calcium in T cell Mitogenesis

In T lymphocytes stimulated by concanavalin A (Con A), interleukin 2 (IL-2) acts on the late G₁ stage of the cell cycle. Ca²⁺ uptake by T cells was not enhanced with the stimulation of Con A (initiation) or IL-2 (late G₁ stage), but Ca²⁺ requirement was observed at the two stages. These results indicate that the enhancement of Ca²⁺ uptake is not necessary, but intracellular Ca²⁺ may act as an important messenger in T cell mitogenesis.     

Phillygenin exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes

Context: Phillygenin (PHI) is an intestinal metabolite of phillyrin from the genus Forsythia. Although the regulatory activity of Forsythia on immune system has been investigated, the effect of PHI on activated lymphocytes is poorly understood.

Objective: This study was aimed to discuss the possible anti-inflammation potential of PHI on mitogen-activated stimulated lymphocytes in vitro.

Methods: Mice spleen lymphocytes were incubated with PHI for 4?h, and then stimulated with concanavalin A (Con A) or phorbol 12-myristate 13-acetate/ionomycin (PMA?+?Ion). Cell viability was assayed by cell counting kit-8 (CCK-8). The expression of CD69 and CD25, proliferation, cell cycle, intracellular Ca²⁺ concentration, apoptosis, mitochondrial inner membrane potential (ΔΨm), mitochondrial permeability transition (MPT), interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were analyzed by flow cytometry. The expression of cyclin B1, cyclin D1, Cyclin E, and the phosphorylation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 were assayed by western blotting.

Results: The results showed that PHI inhibited the proliferation of Con A-activated lymphocytes and induced a G₀/G₁ phase arrest by suppressing cyclin D1 and cyclin E. Meanwhile, PHI antagonized Con A-induced T cells activation through blocking intracellular Ca²⁺ overload and suppressing the phosphorylation of JNK and Erk1/2. Both Con A and PMA?+?Ion-induced secretion of IL-2, IFN-γ, and TNF-α were attenuated by PHI. PHI enhanced Con A-induced lymphocytes apoptosis through decreasing ΔΨm and increasing MPT.

Conclusion: These results suggest that PHI exhibits its anti-inflammatory activity through modulating multiple cellular behaviors, leading to the suppression of the adaptive immune response.     

Experimental diabetes attenuates calcium mobilization and proliferative response in splenic lymphocytes from mice

The present study was conducted to investigate the effects of the diabetic condition on cytosolic free Ca²⁺ concentration, [Ca²⁺]_i, and the proliferation of splenic lymphocytes from mice. Diabetes was induced in mice by intraperitoneal injection of alloxan. [Ca²⁺]_i and the proliferation ex vivo of splenic lymphocytes isolated from mice were examined using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively. Diabetes caused a significant increase in resting [Ca²⁺]_i and significantly reduced the ability of concanavalin A (Con A; a T-lymphocyte-selective mitogen) to increase [Ca²⁺]_i, but not that of lipopolysaccharide (LPS; a B-lymphocyte-selective mitogen). In addition, diabetes significantly reduced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Verapamil (an L-type Ca²⁺ channel blocker) inhibited Con A-induced increases in [Ca²⁺]_i and proliferation in lymphocytes from control and diabetic mice to a similar extent, respectively. These results suggest that diabetes attenuates Con A-stimulated T-lymphocyte proliferation by decreasing [Ca²⁺]_i via reduction of Ca²⁺ entry through L-type Ca²⁺ channels.     

Marine peptides as immunomodulators: Californiconus californicus-derived synthetic conotoxins induce IL-10 production by regulatory T cells (CD4+Foxp3+)

Abstract

Context: CD4⁺ T lymphocytes are able to differentiate into distinct subtypes according to several immunological scenarios, including T helper (Th)1, Th2, Th17 and regulatory T (Treg) cells. CD4⁺ T cells are phenotypically flexible and have specific ion channels, such as the nicotinic acetylcholine receptors (nAChR) that could be modulated by peptides produced by marine snails, known as conotoxins. Their effect on T lymphocytes has not been explored and emerging evidence suggests that these peptides may have immunomodulatory activities.

Objective: This study investigated the effect of two Californiconus californicus-derived synthetic conotoxins on the proliferation and differentiation of T lymphocyte subpopulations Th1, Th2, Th17 and Treg.

Methods: Cells from lymph nodes of BALB/c mice were cultured in the presence of conotoxins cal14.1b and cal14.2c (5.5?μM), during 96?h. Cell proliferation and intracellular cytokine production (IFN-γ, IL-4, IL-17 and IL-10) were analyzed by flow cytometry.

Results and Discussion: cal14.1b and cal14.2c increased intracellular IL-10 production in Treg (CD3⁺CD4⁺Foxp3⁺) cells and decreased intracellular IL-17 production (CD3⁺CD4⁺) after 72?h of culture. Conotoxins did not show any effect on T cell proliferation nor Th1/Th2 balance.

Conclusion: These results suggest that synthetic conotoxins exert immunomodulatory activity, especially by regulating specific functions on T lymphocytes.     

A T cell activation antigen,Ly6C,induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8⁺ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8⁺ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C⁺ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G₀/G₁ to S+G₂/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4⁺ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4⁺ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Immunomodulatory effects of the pentapeptide YGSRS on human peripheral-blood mononuclear cells

Abstract

Context: The pentapeptide YGSRS is originated from coffee bean, while its pharmacological features have little been examined.

Objectives: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+ CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells (PBMCs) activated by T-cell mitogen.

Materials and methods: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessed by WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture medium were analyzed with beads-array procedures followed by analysis with flow cytometry. The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry.

Results: YGSRS at 1–10?000?ng/ml (1.56–15?600?nM) has a tendency to promote the mitogen-activated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affect the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, IL-6 and IL-10 from the activated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in the medium were observed at 1–1000?ng/ml (1.56–1560?nM) (p?<?0.05). YGSRS has a tendency to decrease the frequency of Treg cells in the activated PBMCs, but the difference was not statistically significant.

Discussion and conclusions: The data suggest that the pentapeptide YGSRS affects the production of several types of cytokines from activated human peripheral T cells, which may modulate Th2 type immunity.     

The anti-inflammatory effect of the SOCC blocker SK&F 96365 on mouse lymphocytes after stimulation by Con A or PMA/ionomycin

SK&F 96365, 51-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H-imidazole hydrochloride, has emerged as a useful pharmacological tool in the study of store-operated Ca²⁺ entry (SOCE). But the precise molecular mechanism and effect of SK&F 96365 on mouse lymphocytes are still not well determined. This study investigated the pharmacological profile of SK&F 96365 on mouse lymphocytes stimulated by mitogen concanavalin A (Con A) or by a combination of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and a calcium ionophore, ionomycin in vitro. Our results showed that SK&F 96365 pre-treatment diminished the cytosolic calcium rise on lymphocytes induced by ionomycin, PMA/ionomycin, and thapsigargin (TG), respectively. CFDA-SE staining results showed that SK&F 96365 (5-20 μM) inhibited both Con A- and PMA/ionomycin-induced lymphocytes proliferation in a time- and dose-dependent manner. Upon the same stimulation, SK&F 96365 inhibited the expression of CD69 and CD25 on CD3⁺ T lymphocytes in a dose-dependent manner. The cell cycle analyzing results showed that SK&F 96365 caused a G0/G1 phase cell cycle arrest on both Con A- and PMA/ionomycin-activated lymphocytes in a dose-dependent manner. In addition, SK&F 96365 induced a decrease in mitochondrial membrane potential (ΔΨm) and promoted mitochondrial permeability transition (MPT) in both Con A- and PMA/ionomycin-activated lymphocytes. Furthermore, SK&F 96365 significantly inhibited the production of proinflammatory cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)), and the anti-inflammatory cytokine (IL-10) on both Con A- and PMA/ionomycin-activated lymphocytes. SK&F 96365 did not induce a statistically significant increase in levels of proinflammatory IL-6 and monocyte chemoattractant protein-1 (MCP-1) but of IL-12p70 upon the stimulation of Con A, whereas these three cytokines were markedly inhibited by it upon the stimulation of PMA/ionomycin. This finding revealed that SK&F 96365 exhibited an anti-inflammatory effect on mouse lymphocytes both upon the stimulation of Con A and PMA/ionomycin, and the precise mechanism of SK&F 96365 inhibiting Con A-activated lymphocytes proliferation is different from PMA/ionomycin.     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

Paeoniflorin suppresses IL-33 production by macrophages

Abstract

Objective: Interleukin (IL)-33 has been attracting more and more attention as a new member of theIL-1 cytokine family in recent years. However, the underlying mechanisms referred to the regulation of endogenous IL-33 production are not fully illustrated. Paeoniflorin (PF) has been reported to possess multiple pharmacological activities, including anti-inflammation and anti-allergy. In this study, we aimed to investigate the effect of PF on IL-33 production by macrophages and explore the underlying mechanisms.

Methods: In vivo, IL-33 production in mice after lipopolysaccharide (LPS) injection together with PF application was detected by enzyme-linked immunosorbent assay (ELISA). In vitro, MTT, Real-time PCR, ELISA, Calcium (Ca²⁺) imaging and Western blot were used to assess the cytotoxicity of PF, IL-33 expression at mRNA and protein levels, Ca²⁺ influx, protein kinase C (PKC) activity, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) activation in LPS-stimulated RAW264.7 macrophages with PF administration.

Results: Our results indicated that PF (5 and 25?mg/kg) significantly reduced the production of TNF-a, IL-1β, and IL-33 in the peritoneal exudate of LPS-treated mice. In vitro assay, upregulation of PF concentration (≥ 20?μM) showed an increased cytotoxicity in RAW264.7 cells during the 24-h cell culture. PF (10?μM) inhibited IL-33 production, Ca²⁺ influx, PKC activity, NF-κB (p65) activation, and P38MAPK phosphorylation in LPS-treated macrophages. Notably, NF-κB inhibitor (BAY 11-7085), P38MAPK inhibitor (SB203580), and Ca²⁺ blocker (NiCl₂) also curbed LPS-induced IL-33 production, respectively.

Conclusions: PF suppresses IL-33 production by macrophages via inhibiting NF-κB and P38MAPK activation associated with the regulation of Ca²⁺ mobilization.     

Human CD4+ effector T lymphocytes generated upon TCR engagement with self-peptides respond defectively to IL-7 in their transition to memory cells

The peripheral repertoire of CD4⁺ T lymphocytes contains autoreactive cells that remain tolerant through several mechanisms. However, nonspecific CD4⁺ T cells can be activated in physiological conditions as in the course of an ongoing immune response, and their outcome is not yet fully understood. Here, we investigate the fate of human naive CD4⁺ lymphocytes activated by dendritic cells (DCs) presenting endogenous self-peptides in comparison with lymphocytes involved in alloresponses. We generated memory cells (Tmem) from primary effectors activated with mature autologous DCs plus interleukin (IL)-2 (Tm_auto), simulating the circumstances of an active immune response, or allogeneic DCs (Tm_allo). Tmem were generated from effector cells that were rested in the absence of antigenic stimuli, with or without IL-7. Tmem were less activated than effectors (demonstrated by CD25 downregulation) particularly with IL-7, suggesting that this cytokine may favour the transition to quiescence. Tm_auto and Tm_allo showed an effector memory phenotype, and responded similarly to polyclonal and antigen-specific stimuli. Biochemically, IL-7-treated Tm_allo were closely related to conventional memory lymphocytes based on Erk-1/2 activation, whereas Tm_auto were more similar to effectors. Autologous effectors exhibited lower responses to IL-7 than allogeneic cells, which were reflected in their reduced proliferation and higher cell death. This was not related to IL-7 receptor expression but rather to signalling deficiencies, according to STAT5 activation These results suggest that ineffective responses to IL-7 could impair the transition to memory cells of naive CD4⁺ T lymphocytes recognizing self-peptides in the setting of strong costimulation.     

IL-38, a potential therapeutic agent for lupus,inhibits lupus progression

Background

Previous studies reported that IL-38 was abnormally expressed in patients with systemic lupus erythematosus (SLE). However, the involvement of IL-38 in the pathophysiology of SLE remains unknown.

Methods

The therapeutic potential of IL-38 was tested in pristane-treated wild-type (WT) and IL-38^?/? mice. Thus, SLE was induced via pristane in WT and IL-38^?/? mice. Afterwards, the liver, spleen, and kidney of each mouse were obtained. The flow cytometric analysis of the immune cells, serologic expression of inflammatory cytokines and autoantibodies, renal histopathology, and inflammatory signaling were evaluated.

Results

WT mice with pristane-induced lupus exhibited hepatomegaly, splenomegaly, severe kidney damages, increased lymphoproliferation, enhanced lymphoproliferation, and upregulated inflammatory cytokines, such as IL-6, IL-13, IL-17A, MIP-3α, IL-12p70, and IFNγ, and elevated levels of autoantibodies, such as ANA IgG, anti-dsDNA IgG, and total IgG. IL-38^?/? mice whose lupus progressed, had elevated cells of CD14⁺, CD19⁺, CD3⁺, and Th1, upregulated inflammatory cytokines and autoantibodies, and severe pathological changes in kidney. Administration of recombinant murine IL-38 to pristane-treated IL-38^?/? mice improved their renal histopathology, which depended on ERK1/2, JNK1/2, p38, NF-κB p65, and STAT5 signaling pathways.

Conclusion

IL-38 regulates SLE pathogenesis. Furthermore, targeting IL-38 is critical in the treatment of SLE.

     

T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed TH1 /TH2 pattern of cytokines

Background: Nasal polyps (NPs) are inflammatory reactions in the nasal mucosa the etiology and pathogenesis of which remains unknown. Objective: The purpose of this study was to study in detail the phenotype and function of T lymphocytes infiltrating NPs by analyzing the expression of surface markers and cytokine secretion. Methods: NP tissue samples and peripheral blood were obtained from 18 patients. Mononuclear cells were purified from these samples, and their phenotype was investigated by triple-color immunofluorescence and flow cytometric analysis. Cytokine production was determined in cultures by using an ELISA technique. Results: NP lymphoid cells mainly consisted of T lymphocytes. These T lymphocytes showed a CD45RO+CD45RA– phenotype and expressed pan-T cell molecules; the CD8+ subset was predominant. NP T cells showed a lower density of CD28, CD3, and TCR-αβ compared with T cells from peripheral blood. NP T lymphocytes expressed the activation markers DR and CD69 and exhibited the adhesion molecule profile CD54+, CD62L–, and CD103+ CD49d^low . Virtually all NP T cells bore CD95 (FAS), but they did not undergo apoptosis, either spontaneously or induced by CD95 cross-linking with the mAb CH11. The pattern of cytokines secreted by NP T lymphocytes was characterized by the spontaneous and simultaneous production of IFN-γ and IL-5. Neither IL-2 nor IL-4 were detectable in nonstimulated cultures. Conclusion: This study defines the T lymphocytes that infiltrate NPs as memory T cells in an activated status, with homing properties related to the mucosal immune system. They are resistant to anti-CD95-mediated apoptosis and produced a mixed T_H1 /T_H2 cytokine pattern as defined by the simultaneous production of IFN-γ and IL-5. (J Allergy Clin Immunol 1998;102:953-60.)     

TGF-β2 and PGE2 in Rabbit Blastocoelic Fluid Can Modulate GM-CSF Production by Human Lymphocytes

PROBLEM: During normal pregnancy, major changes occur in the production of Th2/Th1 cytokines at the feto-maternal interface. Th2 cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10) are predominantly produced locally in the uterine and placental tissues, whereas the production of Th1 cytokines such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) are decreased. Because these modulations might be induced by the embryo, the current study was carried out to test the effect of rabbit blastocoelic fluid on the production of Th2/Th1 cytokines by lymphocytes, and to investiate the possible implication of transforming growth factor β₂ (TGF-β₂) prostaglandin E₂ (PGE₂) as modulators of the production of these cytokines. METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were cultured along with ConcanavalinA (Con A), and rabbit blastocoelic fluid was collected on day 12 of gestation (BF d-12). Concentrations of cytokines in culture media were determined by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Addition of BF d-12 in the culture medium induced a strong inhibition of IL-2, TNF-α, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. However, an initial pretreatment of the lymphocytes with BF d-12, followed by a Con A stimulation, led to a marked increase in GM-CSF production, whereas IL-2, TNF-α, and IL-10 secretions were inhibited. It was also demonstrated, for the first time, that a pretreatment of the lymphocytes with TGF-β₂ and PGE₂ increased GM-CSF production to the same level reached after the addition of BF d-12. Furthermore, removal of TGF-β₂ and PGE₂ from BF d-12 by affinity chromatography reduced the effect of BF d-12 on GM-CSF production. CONCLUSIONS: Taken together, these findings suggest that the embryo, in modulating harmful and beneficial cytokine production locally, plays an active role in its protection against maternal immune cellular assault. These results also emphasize the importance of growth factors for successfully maintaining pregnancy.     

Immunomodulatory effects of epicatechin-(2β→O→7, 4β→8)-ent-epicatechin isolated from Rhododendron spiciferum in vitro

Abstract

Context: Many traditional Chinese medicines (TCMs) can act as either immunosuppresants or immunostimulants, properties that have lead to their increasing use as immunomodulators in the treatment of disease. Recently, our lab successfully identified a dimer epicatechin-(2β→O → 7, 4β→8)-ent-epicatechin (EEE) from the chloroform extract of Rhododendron spiciferum.

Objective: To evaluate the immunomodulatory effects of EEE in vitro.

Materials and methods: Splenocytes, peritoneal macrophages and peripheral blood mononuclear cells from BALB/c mice were incubated with different concentrations of EEE.

Results: EEE significantly stimulates splenocyte proliferation when administered either alone or in combination with concanavalin A (Con A), lipopolysaccharide (LPS), and Anti-CD3. EEE enhances the cytotoxicity of natural killer (NK) cells markedly and phagocytic function of macrophage. Moreover, we found that the levels of several helper T1 (Th1) cytokines, including interleukin-2 (IL-2), IL-12, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are significantly increased after EEE treatment, while the levels of Th2 cytokine IL-4 and IL-10 are significantly decreased. As a result, the ratio of Th1/Th2 is significantly increased in the presence of EEE. EEE also increased CD4 and CD8 cell populations.

Conclusion: These results indicate that EEE exhibits immunomodulatory activity and suggests that this compound could be developed as a novel immunotherapeutic agent for treating cancer and other immune-mediated diseases.     

Cytokine patterns in adults with aids

Background: It has been reported that in HIV infected patients enhanced production of IL-4 and IL-10 in response to stimulation of peripheral blood lymphocytes with phytohemagglutinin is associated with disease progression. Some have proposed that a switch from a cytokine profile associated with CD4+ Th1 predominance (IL-2, IFN-G, TNF-B) to Th2 preddominance (IL-4, IL-5) plays a major role in the progression of HIV infection. Others find no clear evidence for the dichotomy of Th1 and Th2 predominance in HIV infected patients Discrepant results have been reported in studied populations in which only a few cytokines have been examined.

Methods: Thirty-one adult patients with AIDS but without other active infections provided serum and penpheral blood lymphocytes for determination of cytokine levels. Their responses were compared to those of five normal healthy volunteers without active infection. ELISA techniques were employed to quanutate cytokine levels. Both circulating lymphocyte and enriched lymphocyte populations were studied with and without stimulation employing phytohemagglutinin and/or rhIL-2.

Results: Patterns of cytokine expression were analyzed in 31 adult patients with AIDS but without other active infections. All had CD4+ cell counts below 50 and large viral loads (Roche PCR). The unstimulated cytokine profile in these patients generally showed marked elevations in IL-I, IL-3, IL-4. IFN-G, TNF-A, TNF-B, OSM, and TGF-B. Minimal elevations compared to normal healthy volunteers were noted for LL-2, IL4, IL-8, IL-12, IFN-A, and IL-6SR. The levels of RANTES were lower than in normal healthy volunteers. Responses of peripheral blood lymphocytes to stimulation with phytohemagglutinin showed enhancement of all cytokines in all subjects studied though the response was much less marked in AIDS patients than in normal volunteers with the exception of IL-3, IL-4, IL-8. TNF-B, and TGF-B whch are increased. Little difference in IL-2 and IL-12 expression was noted between stimulated peripheral blood lymphocytes of AIDS patients and normal healthy volunteers. No relation was noted with patient age or with any use of antiretroviral agents. Recombinant human IL-2 was a less potent stimulant than was phytohemagglutinin.

Conclusion: The character of cytokine response in AIDS patients may be directly related to the stimulus employed in test systems. There is no evidence for Th1/Th2 dysregulation. Cytokine elevations in AIDS patients generally are reflective of chronic infection (the virus). Lymphocytes from AIDS patients do not respond as well to stimulation as do those from normal healthy volunteers. The stimulated lymphocyte response in AIDS patients suggests there is underlying low-grade host versus virus reaction in these patients (exaggerated responses of IL-3, IL-4, IL-8, TGF-B).     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense

Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment. i-IEL comprise unique T cell populations including CD4^-CD8αα⁺ T cells expressing T cell receptor (TCR)αΒ or TCRγδ and CD4⁺ CD8αα⁺ T cells expressing TCR αΒ. We show here that CD4⁺ CD8αα⁺ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway. i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia. The interaction of i-EC and i-IEL through E-cadherin/α_EΒ₇ integrin is important for homing and maintenance of i-IEL in intestine.Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells. Invasion ofL. monocytogenes into i-EC activated NFk-B and subsequently up-regulated the expression of IL-15 gene, which has a NFk-B binding site at the promoter region. i-IEL, especially γδ T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection withL. monocytogenes in mice and rats. The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection. Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine.