CD4+ T Cell-derived IL-10 Promotes Brucella abortus Persistence via Modulation of Macrophage Function

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4⁺CD25⁺ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25⁺CD4⁺ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.     

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

Murine and Bovine �æ� T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ^−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα^−/−, and GMCSF^−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ^−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.     

Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum

By immunohistochemistry, CGRP-like immunoreactive (CGRP-LI) nerve fibres were found in the lamina propria along small vessels and in the lamina muscularis mucosae in the porcine ileum. Immunoreactive nerve cell bodies were found in the submucous and myenteric plexus. Upon HPLC-analysis of ileal extracts, CGRP-LI corresponded entirely to porcine CGRP plus smaller amounts of oxidised CGRP. Using isolated vascularly perfused segments of the ileum, we studied the release of CGRP-LI in response to electrical stimulation of the mixed extrinsic periarterial nerves and to infusion of different neuroblockers. In addition, the effect of infusion of capsaicin was studied. The basal output of CGRP-LI was 2.9+/-0.7 pmol/5 min (mean+/-S.D.). Electrical nerve stimulation (8 Hz) significantly increased the release of CGRP-LI to 167+/-16% (mean+/-S.E.M.) of the basal output (n=13). This response was unaffected by the addition of atropine (10(-6) M). Nerve stimulation during infusion of phentolamine (10(-5) M) with and without additional infusion of atropine resulted in a significant further increase in the release of CGRP-LI to 261+/-134% (n=5) and 240+/-80% (n=9), respectively. This response was abolished by infusion of hexamethonium (3x10(-5) M). Infusion of capsaicin (10(-5) M) caused a significant increase in the release of CGRP-LI to 485+/-82% of basal output (n=5). Our results suggest a dual origin of CGRP innervation of the porcine ileum (intrinsic and extrinsic). The intrinsic CGRP neurons receive excitatory input by parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors. The stimulatory effect of capsaicin suggests that CGRP is also released from extrinsic sensory neurons.     

Vasoactive intestinal polypeptide: increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle

Vasoactive intestinal polypeptide (VIP) was examined $\text{in vitro}$ for effects on tone and neuronal release mechanisms in intestinal smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea pig ileum and rabbit jejunum in concentrations ranging from 10^?9 to 10^?7 M. Increased tone in the guinea pig ileum was partially antagonized by the anticholinergic agent, atropine (4.38 × 10^?6 M) suggesting that one component of the contractile response was due to the indirect release of acetylcholine. The H₁ receptor antagonist, pyrilamine, did not alter the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The ability of VIP to modulate acetylcholine release was confirmed in field stimulated ileal preparations where VIP increased the force developed to endogenously released acetylcholine without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP related cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the field stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the ability of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal patholgies involving excessive mucous secretion and motility.     

The role of the ileum in the control of food intake and intestinal adaptation

Studies on animals that drastically reduce their food intake after having a jejunoileal bypass or an ileal transposition surgery suggest that the lower ileum may play a major role in the control of daily food intake. In this study, eight rats were given slow continuous infusions of either 18, 28, or 38 mL of their normal liquid diet directly into their upper ileum. They reduced their daily intake in a compensatory way for the two smaller infusions and in a more than compensatory way for the large infusion. The later results suggests that the large infusion may have caused the rats some discomfort, which led to a lower food intake. This was tested in a conditioned aversion paradigm with an ileal infusion of 26 mL of the diet into eight naive rats. These rats showed a strong aversion to the ileal infusion. Infusion of the same amount of diet into the stomach of eight other rats failed to demonstrate an aversion and showed that the procedures of the experiment did not produce the aversion. The infusion of relatively small amounts of liquid diet into the ileum produces an internal signal that reduces intake and is regulatory. A second process in which ileal infusion causes discomfort leads eventually to a more than regulatory decrease in daily intake.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

Role of gastrin-releasing peptide in pepsinogen secretion from the isolated perfused rat stomach

We studied the effects of the neuropeptide gastrin-releasing peptide on pepsinogen secretion using an isolated perfused rat stomach with intact vagal innervation. Following electrical stimulation of the vagus nerves, the pepsin output to the luminal effluent increased from 94 ± 7 to 182 ± 24 units pepsin/min and the release of immunoreactive gastrin-releasing peptide to the venous effluent increased from 0.059 ± 0.014 to 0.138 ± 0.028 pmol/min. Infusion of gastrin-releasing peptide at 10⁻⁸ M significantly increased pepsin output (from 87 ± 17 to 129 ± 22 units pepsin/min) and simultaneous infusion of gastrin-releasing peptide and carbachol at 10⁻⁸ and 10⁻⁶ M, respectively, resulted in an increase to almost 4 times the basal values. Atropine reduced but did not abolish the pepsin response to vagal stimulation and to infusion of gastrin-releasing peptide. Our results suggest that gastrin-releasing peptide participates in the vagal control of pepsinogen secretion.     

Do normal plasma neurotensin concentrations increase ileal output?

Infusions of neurotensin increase ileal secretion in experimental animals, and the volume of ileal effluent in patients with ileostomies. The aim of the present study was to determine whether normal postprandial plasma concentrations of neurotensin increase the volume of fluid leaving the ileum. Basal and peak postprandial plasma neurotensin concentrations were 23 (17-36) and 39 (25-43) pmol/l (median and range) respectively in five subjects with ileostomies and 15 (3-27) and 32 (15-82) pmol/l respectively in nine normal subjects. Infusion of neurotensin for 30 min at a rate of 6.3 pmol/kg/min into six patients with ileostomies increased ileostomy output about 10-fold, and produced a significant decrease in the concentration of solid material, but plasma neurotensin concentrations rose to 237 (82-422) pmol/l during infusion at this rate. Infusion of neurotensin at 2.3 pmol/kg/min, producing plasma levels of 60 (16-108), had no significant effect the amount or nature of ileostomy effluent. We conclude that normal postprandial plasma concentrations of neurotensin are unlikely to influence the volume of fluid leaving the ileum.     

Outer Membrane Vesicles from Brucella abortus Promote Bacterial Internalization by Human Monocytes and Modulate Their Innate Immune Response

Outer membrane vesicles (OMVs) released by some Gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa) and monocytes (THP-1), and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8) to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively). Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.     

Lactoferrin-mediated modulation of mononuclear cell activities: I. Suppression of the murine in vitro primary antibody response

The effect of the iron-binding glycoprotein lactoferrin (LF) on the in vitro primary antibody response of mouse splenic cells to a T-lymphocyte-dependent antigen, sheep erythrocytes (SE), and a T-lymphocyte-independent antigen, trinitrophenolated Brucella abortus, was examined. Both iron-saturated and native LF (8% saturated) at 10^?10 to 10^?6M concentrations but not transferrin (an iron-binding glycoprotein similar to LF) significantly (P < 0.05) decreased the number of direct (IgM) plaque-forming cells (PFC) to SE and trinitrophenol (TNP) as determined by the hemolytic plaque assay. LF was equally effective in decreasing the PFC response to TNP in T-lymphocyte-depleted splenic cell cultures. Concentrations of LF which decreased the PFC response were noncytotoxic. A 1-hr exposure of splenic cells to LF at the beginning of the 5-day culture period but not 1 hr prior to assaying for PFC, or exposure of isolated macrophage-rich but not lymphocyte-rich populations to LF prior to reconstitution resulted in a significant decrease in the anti-SE response. These data suggest that LF which is synthesized and released by polymorphonuclear leukocytes and present in inflammatory lesions may play a role in modulating the antibody response through an effect on the macrophage.     

Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial

 Adoptive immunotherapy (AIT) of cancer with T lymphocytes may be limited by the need to activate tumor antigen-sensitized cells in vitro. In murine models, we have shown that AIT with tumor-sensitized T cells that have been pharmacologically activated with bryostatin 1 and ionomycin plus interleukin-2 can induce tumor regression. A Phase I clinical trial was carried out to assess the feasibility and toxicity associated with using tumor- or vaccine-draining lymph node cells, activated pharmacologically and expanded in culture with low-dose interleukin-2 and infused intravenously, followed by IL-2 infusion. Nine patients were entered into the trial, and six were treated as planned. Average expansion of cell numbers over 13 to 27 days in culture was 118-fold. No patient's cells reached the target cell number (2.5 × 10¹⁰). Infusion of these cells did not result in any unexpected toxicities. The toxicities observed were related to IL-2 infusion, and conformed to the expected range of side-effects. Based on these Phase I results, additional trials, with tumor antigen vaccine-sensitized DLN and technical modifications of the culture technique, are planned. Received: 18 January 2001 / Accepted: 26 April 2001     

Tolerance induction to a thymus-dependent antigen in vitro: treatment of nonadherent cells with tolerogen biologically filtered in vitro

Highly tolerogenic bovine gamma globulin (BGG), a thymus-dependent antigen, was prepared by biologic filtraration in vitro. It readily induced tolerance in vivo in BALB/c mice and also rendered their nonadherent lymph node cells tolerant after in vitro incubation. Biologic filtration in vitro was carried out by incubating 2.5 × 10⁷ lymph node cells with 10 mg of nontolerogenic BGG in 10 ml of Eagle's medium containing 2% normal mouse serum at 37 °C for 6 hr. The BGG-containing medium was clarified by centrifugation and was used without further dilution.For tolerance induction in vitro, lymph node cells were separated into adherent and nonadherent populations on Falcon plastic. These cells were incubated for 0–18 hr at 37 °C with biologically filtered BGG (bBGG). After incubation, the cells were washed three times and (2–2.5) × 10⁷ nonadherent or 4 × 10⁶ adherent cells were injected iv with their untreated counterpart into lethally irradiated mice which had received 10⁶ bone marrow cells. The recipients were then challenged with 300 μg of aggregated BGG, and tolerance was assayed by the elimination of labeled BGG, rosette formation, and passive hemagglutination. Spleen cells were similarly treated for comparison. Our findings show that tolerance was not induced in vitro in adherent lymph node cells. However, in the nonadherent populations, those from the lymph node but not the spleen were rendered tolerant. The acquisition of tolerance in vitro was gradual. It was dependent upon the length of exposure to bBGG and required at least 6 hr.     

Brucella abortus induces intracellular retention of MHC‐I molecules in human macrophages down‐modulating cytotoxic CD8+ T cell responses

Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8⁺ T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN‐γ‐induced MHC‐I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC‐I down‐modulation correlated with the development of diminished CD8⁺ cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8⁺ T lymphocytes and a diminished percentage of IFN‐γ‐producing CD8⁺ T cells. Inhibition of MHC‐I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC‐I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC‐I molecules whereby B. abortus would avoid CD8⁺ cytotoxic T cell responses, evading their immunological surveillance.     

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during Brucella abortus infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.     

Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

1. In rat ileal smooth muscle both adenosine and ATP at 10⁻⁴ M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P₁ receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the P₂ receptor antagonist quinidine.2. The P₂ receptor desensitizer α,β-methylene-ATP was without effect on ACh responses nor did the stable analogue β,gg-methylene-ATP exert any effect on spontaneous mechanical activity.3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum.4. ATP potentiated the phasic component of the ileal K-induced contracture but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force.5. α,β-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. β, γ-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force.6. The results of this study suggest that rat ileum may contain the A₁ subtype of the P₁ receptor but the evidence for a P₂ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine.7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.     

Brucella abortus induces apoptosis of human T lymphocytes

Immune evasion is essential for Brucella abortus to survive in the face of robust adaptive CD4+ T cell response. We have previously demonstrated that B. abortus can indirectly inhibit CD4+ T cells by down-regulating MHC-II expression and antigen presentation on macrophages. However, whether B. abortus is able to directly interfere with T lymphocytes is not known. We report here that B. abortus induces apoptosis of human T lymphocytes, even though invasion of T lymphocytes was low and non-replicative. The ability of heat-killed B. abortus to reproduce the same phenomenon suggested that there was a bacterial structural component involved. We demonstrated that a prototypical B. abortus outer membrane lipoprotein (l-Omp19), but not its unlipidated form, induced T lymphocyte apoptosis. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also induced an increase in T lymphocyte cell death, indicating that the structural component implicated in the phenomenon could be any B. abortus lipoprotein. B. abortus-induced T lymphocyte apoptosis was dependent on the secretion of TNF-α since pre-incubation of T lymphocytes with anti-TNF-α mAb inhibited the apoptosis of the cells. Overall, these results represent a new mechanism whereby B. abortus by directly inhibiting T cell-mediated responses may evade adaptive immune responses.     

Influence of exogenous xylanase supplementation on apparent metabolisable energy and amino acid digestibility in wheat for broiler chickens

The influence of two commercial xylanase preparations on the apparent metabolisable energy (AME) and, ileal and excreta amino acid digestibilities in wheat for broilers was investigated. The following three dietary treatments were tested: a basal diet containing 918 g kg⁻¹ wheat, basal diet plus enzyme 1 and basal diet plus enzyme 2. Each diet was offered ad libitum to four pens (four birds/pen) from day 35 to day 42 post-hatching. Total collection of excreta was carried out during the last 4 days. All birds were euthanatised on day 42 and the contents of the lower half of the ileum were collected. Apparent ileal and excreta amino acid digestibilities were calculated using acid-insoluble ash as the indigestible marker. The AME values of wheat were markedly improved by exogenous enzymes. Addition of enzymes 1 and 2 improved the AME values by 12.6% and 18.6%, respectively. Supplementation of enzymes resulted in significant (p<0.05) improvements in ileal and excreta amino acid digestibilities in wheat. Mean excreta digestibility coefficients of the 15 amino acids in wheat, wheat plus enzyme 1 and wheat plus enzyme 2 were 0.70, 0.78 and 0.79, respectively. The corresponding values at the ileal level were 0.78, 0.84 and 0.85, respectively. Excreta digestibility of amino acids, with the exception of tyrosine, were found to be consistently lower than the ileal estimates. In the case of unsupplemented wheat, apparent digestibilities of individual amino acids measured in excreta were 5–22% units lower (p<0.10–0.01) than those measured at the terminal ileum. The ileal-excreta differences were generally lowered by the addition of enzymes.     

Dissociation of in vitro lymphocyte transformation from production of a mononuclear leucocyte chemotactic factor in agammaglobulinemic chickens

Cultures of splenic and peripheral lymphocytes from normal chickens immunized intravenously with Brucella abortus organisms were stimulated by this antigen to incorporate significantly greater amounts of ³H-thymidine and ¹⁴C-leucine than lymphocytes from unimmunized animals. Lymphocytes from immunized agammaglobulinemic chickens were unresponsive to Brucella. This defect could not be corrected by the addition of either normal nonimmune or irradiated normal immune spleen cells to cultures of ag chicken lymphocytes which suggests that normally B cells transform in vitro in response to this antigen. In contrast, cultured peripheral blood leucocytes from both immunized normal and agammaglobulinemic chickens produce significantly more monocyte chemotactic factor in response to Brucella than leucocytes from nonimmune chickens. This indicates that the production of this mediator is a B cell independent function and suggests that T cells are the producers of this lymphokine.