Mitogen-stimulated Lymphocytes Research Articles

Adipogenic differentiation alters the immunoregulatory property of mesenchymal stem cells through BAFF secretion

Although it has been widely demonstrated that mesenchymal stem cells (MSCs) exert potent immunosuppressive effect, there is little information as to whether adipogenic-differentiated MSCs (adi-MSCs) share the same property. Here, adi-MSCs enhanced alloantigen or mitogen-stimulated lymphocyte proliferation, whereas undifferentiated MSCs (ud-MSCs) inhibited the proliferation. Transwell experiment showed that the stimulatory effect of adi-MSCs was cell–cell contact-independent, and required soluble factors. Furthermore, the supernatant of cultured adi-MSCs could effectively costimulate T and B-lymphocyte proliferation and activation in the presence of anti-CD3 and anti-mu chain treatment, respectively. Production of cytokines interferon-gamma and tumor necrosis factor-alpha by T cells, and Ig secretion by B cells also were increased by the supernatant of cultured adi-MSCs. Mechanism conducted showed that the mRNA and protein expression of costimulatory molecule B-cell activating factor (BAFF) was upregulated, and soluble BAFF was secreted in MSCs after adipogenic differentiation. By blocking the BAFF molecule with specific monoclonal antibody in the culture, T and B-lymphocyte proliferation and activation was stimulated by adi-MSCs or the supernatants were greatly reduced. In conclusion, adipogenic differentiation may alter the immunoregulatory property of MSCs, leading to stimulation of lymphocytes response. The BAFF molecule secreted by the adi-MSCs was responsible for this event.

Micronucleus evaluation in mitogen-stimulated lymphocytes of patients with acromegaly

Acromegaly is a syndrome characterized by a sustained elevation of circulating growth hormone and insulin-like growth factor-1 (IGF-1). Insulin-like growth factor-1 is a potent mitogen and has a role in the transformation of normal cells to malignant cells. This study aims to evaluate the spontaneous micronucleus (MN) frequency by using the cytokinesis-block MN assay to determine genetic damage in the lymphocytes of patients with acromegaly. The study was carried out in 20 patients who had active acromegaly and in 20 age- and sex-matched healthy controls. The MN values were measured in binucleated cells obtained from mitogen-stimulated lymphocytes of patients and control subjects. The distribution of binucleated cells with 1, 2, 3, or more MNs was also measured. We found significantly higher MN frequency values in the lymphocytes of acromegalic patients than in those of the control subjects (2.23 ± 0.68 vs 1.03 ± 0.54, P = .001). The MN frequency increased with increasing IGF-1 levels of acromegalic patients (P = .036, R = 0.47). We observed that the number of binucleated cells with 2 MNs was higher for the majority of patients with acromegaly than for control subjects. Furthermore, the receiver operating characteristic curve (area under the curve = 0.914, P < .0001) was calculated to assess the discriminative power of the MN frequency. Our results indicate that increased MN frequency in the lymphocytes of patients with acromegaly may reflect genomic instability and this increased MN frequency may be associated with elevated levels of circulating growth hormone and IGF-1.

RANKL expression in human T-lymphocytes requires cooperative signaling through the T-cell receptor and adhesion molecule CD2

T-lymphocytes contribute to osteolysis in rheumatic diseases through their production of the osteoclastogenic cytokine RANKL. Mitogenic stimulation of lymphocytes has been shown to induce expression of RANKL; however, the extracellular events leading to its production have not been identified.

Long-term regulation of Na,K-ATPase pump during T-cell proliferation

The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

In vitro Effects of L-Ascorbic Acid and Acrylamide on Lymphocyte Proliferation in Young and Aged Mice

This study examined the effects of Acrylamide (ACR) and L-ascorbic acid (AsA) on the proliferation of splenocytes and the mitogen-stimulated lymphocyte proliferation in young (8 weeks) and aged (82 weeks) C57BL/6 male mice in vitro. AsA increased splenocyte proliferation in both groups; however, this effect was higher in old mice, while the proliferation of lymphocyte was decreased except for treatment at 1 ㎍/㎖ low concentration in both mice. In addition, ACR treatment resulted in decreased LPS-induced B lymphocyte proliferation and ConA-induced T lymphocyte proliferation in both groups. However, AsA increased LPS/ConA-induced lymphocyte proliferation in young groups and had no effects in old mice except at 0.5 ㎍/㎖. Thus, the present data indicate that there is no difference effect of ACR and AsA on lymphocyte proliferation, whereas the effect of AsA on mitogen-induced cell proliferation was reduced in old mice. Overall, our results suggest that various immunomodulators have differing effects of lymphocytic proliferation on young versus aged mice.

Alteration of lymphocyte opioid receptors in methadone maintenance subjects

Methadone maintenance therapy is the most widely used treatment in patients with heroin addiction. Multiple studies have suggested that both current and former heroin addicts entering a methadone maintenance treatment program have altered immune function. Our previous study indicated that heroin addicts have depressed mitogen-stimulated lymphocyte proliferation and a decrease in the modulation of lymphocyte surface markers. This immunosuppression may be mediated via the direct interaction of opiates with lymphocyte opioid receptors. In order to test this hypothesis, the levels of opioid receptors on immune cells obtained from heroin users were determined using saturation binding, and it was found that former heroin addicts on methadone maintenance treatment had a significantly reduced maximum number ( B max) of [ 3H]naloxone binding. The B max values were 51.3 ± 7.6 fmol/mg protein for the non-addicted group and 25.3 ± 3.1 fmol/mg protein for the methadone maintenance group. Opioid receptor gene expression on the immune cell was determined using a semi-quantitative reverse-transcription polymerase chain reaction technique with specific pairs of primers to amplify mu- and delta-opioid receptor mRNAs. Both types of mRNAs were significantly decreased in lymphocytes obtained from the former heroin addicts on methadone maintenance subjects. Similarly, in an in vitro study, 100 μM methadone significantly down-regulated both mu- and delta-opioid receptor mRNA expressions in cultured lymphocytes obtained from naïve subjects. This effect was prevented by including 100 μM naloxone or pretreating with 50 ng/ml pertussis toxin. The data presented indicate that chronic opiate exposure was associated with down-regulation of G-protein-coupled opioid receptor gene expression in human lymphocytes.

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The increase in cholesterol synthesis following mitogen stimulation of mouse lymphocytes was accompanied by a similar rise in dolichol + dolichyl acyl ester synthesis. The ratio of dolichol to cholesterol synthesis did not change during the 24 h period following mitogen stimulation. No synthesis of dolichyl phosphate was detected in mitogen-stimulated lymphocytes.

Cytometric detection of chromatin relaxation, an early reporter of DNA damage response

One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.

Mobiloization of intracellular calcium ions in chicken and rat lymphocytes induced by T cell mitogens

Cytosolic Ca(2+) is known to be an important factor in intracellular signaling pathways that regulate several cellular functions. The present study was designed to measure the intracellular concentrations of Ca(2+) ([Ca(2+)](i)) in T cell mitogen-stimulated chicken lymphocytes, and to compare the results with those in rat lymphocytes. [Ca(2+)](i) was increased in the thymocytes, splenocytes and bursacytes of chickens, and in the thymocytes and splenocytes of rats following exposure to the mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Increases were greatest in the thymocytes followed by the splenocytes and bursacytes. The PHA-induced changes in the thymocytes and splenocytes were similar in chickens and rats, but the ConA-induced increases were significantly lower in the chickens than rats. Pretreatment with EGTA before the application of PHA and ConA completely suppressed the rise in [Ca(2+)](i) in all the chicken lymphocytes, indicating that the increases that occurred in PHA- and ConA-treated chicken lymphocytes could be entirely attributed to the influx of extracellular Ca(2+). On the other hand, the PHA- and ConA-induced increase in [Ca(2+)](i) in rat lymphocytes was not completely suppressed by EGTA, indicating the recruitment of Ca(2+) from the intracellular Ca(2+) pool. The results suggest species differences in the Ca(2+)-based responses to T cell mitogens between chicken lymphocytes and rat lymphocytes.

Biological Properties and Characterization of Lectin from Red Kidney Bean (Phaseolus Vulgaris)

Red kidney beans (Phaseolus vulgaris) contain significant amounts of lectins which have both beneficial and detrimental biological properties. Lectins are carbohydrate-binding glycoproteins that can react specifically with human blood cells, preferentially agglutinate malignant cells, and undergo mitogenic stimulation of lymphocytes. Some lectins are resistant to heat and proteolytic enzymes and can enter the circulatory system intact. Phytohaemagglutinin (PHA)—a lectin isolated from the red kidney bean—consists of four subunits with a molecular weight of 125 kDa. This bioactive compound has been partially purified by affinity chromatography using Affi-gel Blue. PHA has been shown to inhibit the viral enzymes, immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and α- and β-glucosidases. This paper will review the chemical properties, biological activity, distribution, isolation, and heath benefits of red kidney bean lectin.

Inhibitory effect of mesenchymal stem cells on lymphocyte proliferation

We investigated the mechanism underlying the inhibitory effect of rat mesenchymal stem cells (MSCs) on non-specific mitogen-stimulated lymphocytes (LCs) and lymphoblasts (LBs). We used MSCs of passages 2-8 prepared from Sprague-Dawley (SD) rats. LCs were isolated from the spleens of SD rats. Mixed LCs reactions of mitomycin C-treated MSCs with concanavalin A (ConA)-stimulated LCs or LBs were performed, and the proliferation inhibition effect was tested by MTS assay. The cytotoxicity of MSCs against naïve and ConA-stimulated LBs was detected, after co-culturing for 24 h, by lactate dehydrogenase release assay. The rate of apoptosis of ConA-stimulated LBs was measured by flow cytometry after incubation with MSCs for 9 h in the ratio 10:1. The MSCs were treated with Fas ligand (FasL), transforming growth factor (TGF)-beta, and interleukin (IL)-10 blocking antibodies and co-cultured with ConA-stimulated LBs to observe the apoptosis and growth inhibitory effect. The main outcomes were bone marrow-derived adherent CD29+, CD44+, CD45-, CD54+, CD95+, and SH-2+ MSCs. FasL, TGF-beta, and IL-10 production by MSCs were visualized by immunocytochemical analysis. MSCs exhibited a dose-dependent growth inhibitory effect on ConA-stimulated LCs and LBs. When treated with anti-FasL and anti-IL-10 blocking antibodies, the inhibitory effect of MSCs on LBs proliferation, and the effect of apoptosis induction on LBs decreased. Anti-TGF-beta blocking antibody treatment did not significantly influence MSCs. Therefore, the inhibitory effects of MSCs against activated LBs were significantly stronger than that against naïve LCs. FasL and IL-10, rather than TGF-beta, play important roles in the immunosuppressive effects of MSCs.

An in vitro study of liposomal curcumin: Stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells

Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFκB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640 + 10% FBS (pH 7.4, 37 °C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640 + 10% FBS. We looked at the toxicity of non-drug-containing liposomes on 3H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol = 7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.

Surface expression of adenosine deaminase in mitogen-stimulated lymphocytes.

Adenosine deaminase (ADA) expression on the surface of mitogen-stimulated lymphocytes was studied by flow cytometry. The gate for lymphocytes was located by cell size (forward scatter), cytoplasmic complexity (side scatter) and by expression of the markers CD2, CD4, CD8 and CD19. After mitogenic proliferation two populations appeared, one corresponding to non-stimulated cells, and the other consisting of larger cells which showed relatively high expression of adenosine deaminase on their surface. The increase was similar to that observed for CD71 expression, and paralleled the increase in 3H-thymidine incorporation. There was a correlation between ADA and CD71 expression (r = 0.92 for phytohaemagglutinin (PHA) and 0.97 for pokeweed mitogen (PWM)). These results suggest a role for ecto-adenosine deaminase in lymphocyte proliferation and/or triggering.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients.

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1 ± 5.7% by flow cytometry and 27.1 ± 6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.

Are progenitor cells pre‐programmed for sequential cell cycles not requiring cyclins D and E and activation of Cdk2?

Based on studies of unicellular organisms or cultured mammalian cells, the generally accepted model of cell-cycle regulation has been developed in which sequential (scheduled) expression of cyclins D, E, A and B and activation of Cdk2 and Cdk1 takes place. It is assumed that the same model is applicable both in vivo and in vitro. In the present study, we compared proliferating marrow cells freshly isolated from healthy individuals with proliferating lymphocytes in cultures. We demonstrate that during progression of freshly collected human bone marrow cells through G(1), S and G(2)/M, only Cdk1 combined with cyclins A and B(1) was distinctly present and active, and its activity gradually increased. In contrast, in vitro growing mitogen-stimulated lymphocytes had perfectly scheduled sequential expression of all four cyclins and Cdk1 and Cdk2 activities. Our findings demonstrate that the pattern of cyclin expression and Cdk activity in bone marrow in vivo is distinctly different from the one observed for normal cells in vitro. Because proliferating bone marrow cells are predominantly expanding populations of committed progenitors, it is likely that during the expansion phase their cell-cycle progression is pre-programmed, being driven solely by Cdk1 combined either with cyclin A or with cyclin B(1). Expansion of progenitor cells thus may not require the early steps of cell-cycle regulation, associated with triggering progression by availability of growth factors and mitogens.

Blood metal levels and metal-influenced immune functions of harbour seals in captivity

Immunological blood parameters and the effects of environmental pollutants on the immune system are important to assess the health status of seals. Animals living permanently in seal centres are useful for development and validation of diagnostic tools for free-ranging animals. In this study, parameters of cellular immunity as well as metal concentrations in blood and metal influence on cell proliferation of seven seals from a seal centre were investigated repeatedly using multi-element analysis and a lymphocyte proliferation assay. The metal concentrations, except for tin and chromium, were in general comparable to those of free-ranging animals of the North Sea. The unstimulated and mitogen-stimulated lymphocyte proliferation showed strong intra- and inter-individual variability, which reflected variability in activation of the immune status. Furthermore, both immunosuppressive and stimulative influences of metals on lymphocytes were found. Summarising, the methods used in this investigation provided useful information on these animals, and their application to free-ranging animals can be recommended.

Photopheresis in HIV-1 Infected Patients Utilizing Benzoporphyrin Derivative (BPD) Verteporfin and Light

An ex vivo trial utilizing photopheresis with Benzoporphyrin Derivative as the photoactive compound, identified the minimum energy levels of light and concentrations of BPD that eradicated both cell-free and cell-associated HIV-1 infectivity without destroying the virus particles or infected leukocytes. Leukocytes remained viable with altered chemokine/cytokine expression. Apoptosis was induced in a minority of CD4 but not CD8 positive cells with a statistically significant increase in cytolytic T-cell activity. In the 24 week clinical trial in 7 HIV-1 infected patients, three who had rapidly rising viral loads prior to initiating therapy stabilized. Two had a sustained greater than 0.5 log decrement and 5 had stable plasma viral loads (less than a 0.5 log increment or decrement) with varied effects on absolute CD4 and CD8 positive lymphocytes counts. One achieved a greater than 1 log decrement in HIV-1 plasma viral load and undetectable in vivo cell-free and cell-associated HIV-1 infectivity with an increased in vitro lymphocyte mitogen stimulation index. Under amended protocol, 5 additional 12 month courses were administered to three additional patients and two of the previous enrollees. Area under the curve for viral load showed a significant decrease from pre- to post-therapy (p 0.007). No associated toxicities were observed.

Basal level micronucleus frequency in stimulated lymphocytes of untreated patients with leukemia

Structural chromosomal aberrations have been described in various types of human leukemia. The micronucleus technique provides a measure of both chromosome breakage and chromosome loss. The present study investigated micronucleus (MN) frequency in mitogen-stimulated peripheral blood lymphocytes from 20 newly diagnosed and untreated leukemia patients: 4 with acute lymphoblastic leukemia (ALL), 10 with acute myeloid leukemia (AML), and 6 with chronic lymphocytic leukemia (CLL).The mean MN frequency for untreated patients was 3.65 ± 1.47 in ALL, 3.55 ± 1.24 in AML, 3.03 ± 1.05 in CLL. No differences in MN frequency were seen between leukemia types ALL, AML, and CLL ( P = 0.503). The mean basal MN frequency for all patients, regardless of leukemia type, was 3.41 ± 1.19, which was significantly higher ( P = 0.001) than that of 20 age-matched control subjects, 1.87 ± 0.75. Although no significant relationship was found between age and MN frequency in patients with leukemia ( r = 0.050; P = 0.835), the MN frequency in the lymphocytes of healthy control increased regularly and significantly with age ( r = 0.531; P = 0.016). These data indicate that the increased baseline MN frequency in lymphocytes of untreated patients with leukemia may reflect genomic instability or deficiency of DNA repair capacity. MN enhancement in this disease may thus be a consequence of the disease process.

An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as ahomolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naive calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with "natural" Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F (ab')2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.