UDK 619:612.017.11 PROLIFERATION OF NAIVE T LYMPHOCYTES AND T LYMPHOBLASTS IN THE PRESENCE OF TISSUE SPECIFIC MACROPHAGES

In this study the antigen-presenting ability of tissue specific macrophages isolated from bone marrow, spleen, peritoneal cavity and lungs was analyzed. Murine macrophages were isolated by a one-step adherence procedure (for 24 hours) and pretreated with mytomycin C. The antigen-presenting ability of the macrophages was tested in T cell proliferation assays. The ability of macrophages to support antigenspecific proliferation of T lymphoblasts was investigated when sheep red blood cell (SRBC)-specific T blasts were stimulated in vitro by antigen in the presence of different numbers of tissue specific macrophages. On the other hand, the abilities of macrophages to induce proliferation of naïve T cells were analyzed in allogeneic and syngeneic mixed leukocyte reactions (MLRs). It was demonstrated that tissue specific macrophages supported antigen specific proliferation of T lymphoblasts in vitro. They also induced the activation of allogeneic and syngeneic T cells. Increasing the number of macrophages co-cultured with T cells, led to a certain inhibitory effect on T cell proliferation.


INTRODUCTION
Classically, the immune response has been divided into an initial innate and a later adaptive response ŠJr Janeway, 1992¹ and macrophages are included in both parts.As very efficient scavenger cells macrophages phagocytize foreign particles and microorganisms ŠDavis et al., 1998¹, while as antigen-presenting cells they are included in the initial phase of the specific immune response ŠDavis et al., 1998¹.It is well known that dendritic cells are specialized antigen-presenting cells with the greatest potential to stimulate T-lymphocytes ŠMassard et al., 1996¹.Data related to the antigen-presenting ability of macrophages are not consistent as, it was shown that macrophages could either activate ŠLee and Wong, 1982; Davis et al., 1998 Although macrophages originate from a common bone marrow progenitor population ŠVan Furth and Sluiter, 1983¹, resident tissue macrophages display considerable heterogeneity ŠForster and Landly, 1981; Laskin et al. 2001¹.In this study we analyzed the ability of murine tissue specific macrophages to induce T cell proliferation in vitro.The antigen-presenting ability of macrophages from bone marrow, spleen, peritoneal cavity and lungs was tested.Their ability to activate sheep red blood cell (SRBC)-specific T lymphoblasts and/or allogeneic and syngeneic naïve T cells in a mixed leucocyte reaction (MLR) was investigated.
T cell proliferation was found in the presence of macrophages from different sources, with the highest T cell proliferative rate obtained in antigen specific MLRs.An inhibitory effect on T cell proliferation was achieved by increasing the number of macrophages.

MATERIALS AND METHODS
Animals.6-8 week old male or female mice (C57BL/6 and CBA) were obtained from the Medical Military Academy, Belgrade.
Macrophage enrichment procedure.Cell suspensions from bone marrow, spleen, peritoneal cavity and lungs were prepared as previously described ŠZivancevic-Simonovic et al., 2004¹.Following isolation, the cells were centrifuged at 200 x g for 10 min, and suspended in RPMI 1640 medium (Gibco), containing 100 IU/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-glutamine, and 10% fetal calf serum (FCS, Gibco).Macrophage enrichment was achieved by a simpleone-step adherence procedure.Cell suspensions were plated on to 100 mm plastic Petri dishes and incubated at 37 o C in 5% CO 2 (incubator, Hereaus) for 24 hours.Adherent cells were removed from the plastic surface by incubating with PBS containing 0.02% disodium EDTA for 20 min at 4 o C. Cells were washed twice with PBS, resuspended in RPMI 1640 medium and incubated (37 o C, 5% CO 2 ) with mitomycin C (25 µg/ml, Sigma).After 20 min, the cells were washed three times in RPMI 1640 medium and resuspended at the final concentration of 1×10 5 cells/ml.
Antigen and immunization.C57BL/6 mice were immunized with SRBC in the hind foot pads and base of the tail.The first, second, and third injections were applied weekly in complete and incomplete Freund's adjuvant and phosphatebuffered saline (PBS), respectively.
T cell isolation.T lymphoblasts were isolated from the periaortic, popliteal, and inguinal lymph nodes of immunized mice on a nylon wool column ŠKappler and Marrak, 1977¹ 7 days after the last injection.An overnight incubation at 37 o C in a 5% CO 2 atmosphere, in RPMI 1640 medium with 10% FCS, was performed to remove the adherent cells.The nonadherent cells were used for the proliferation assay.
Naïve T cells were isolated by nylon wool filtration ŠKappler and Marrak, 1977¹ of lymph node cells from nonimmunized CBA (for allogeneic MLRs) or C57BL/6 (for syngeneic MLRs) mice.
In vitro cell proliferation assay.T lymphoblasts (4 x 10 5 /well) were stimulated for 96 hrs with 1x10 6 SRBC.The cell proliferation assay was performed in the presence or absence of mitomycin C-pretreated macrophages (1 x 10 3 , 5 x 10 3 or 1 x 10 4 cells/well) in 96-well round-bottom microtiter plates.Allogeneic MLRs were established by adding C57BL/6 macrophages (1 x 10 3 , 5 x 10 3 or 1 x 10 4 cells/well) to 4 x 10 5 CBA lymphocyte (CD3 + ) and syngeneic MLRs by adding C57BL/6 macrophages to T cells isolated from lymph nodes of nonimmunized C57BL/6 mice.The assay was performed in 96-well round-bottom microtiter plates in the presence of indomethacin as stated above.Proliferation was measured by adding 1 µCi (0.037 MBq) Š 3 H¹ thymidine for the last 24 hours of the culture period.Responses were reported as the mean of triplicate tests, counts per minute (cpm) ± SD.
Flowcytometry.For immunofluorescence analysis of CD3, CD4 or CD8 expression on cells isolated by nylon wool filtration, 1x10 6 cells were incubated with rat anti-mouse CD3, CD4 or CD8 MAbs (1 µg/million lymphocytes), for 20 min at 4 o C. Cells were washed three times with PBS, containing 2% FCS, and 0.01% NaN 3 .Following that, the cells were incubated with FITC-labeled goat-anti-rat antibodies (Sigma) (1 mg/assay) for 30 min.Cells were washed three times with PBS, 2% FCS, 0.01% NaN 3 , resuspended in 1% formaldehyde and analyzed on a Flowcytometer equipped with a 488 nm Argon laser light source and a 515 nm band pass filter for FITC-fluorescence.A total of 10 000 events were acquired for analysis using CellQuest software.A histogram plot of FITC-fluorescence (x-axis) versus counts (y-axis) was obtained in logarithmic fluorescence intensity.

RESULTS
It was shown that specific T lymphocytes do proliferate in the presence of antigen and macrophages as antigen-presenting cells.After isolation of lymph node cells, flowcytometry analysis revealed that 96.9% of the nylon wool isolated lymphocytes were CD3-positive (Fig 1).The proportion of CD4+ cells was 65.3% whereas the proportion of CD8+ was 30.7%.When T cells isolated from the lymph nodes of immunized mice were stimulated with three doses of SRBC (1x10 5 , 5x10 5 or 1x10 6 , data not shown) and different concentrations of macrophages, the highest proliferative response was obtained with 1x10 6 SRBC and all following experiments were done in the presence of that antigen dose.
We demonstrated that macrophages (24-hour adherent) isolated from bone marrow, spleen, peritoneal cavity and alveolus were able to present SRBC to specific T lymphoblasts in vitro.Although T cell proliferation was obtained in the presence of all macrophage concentrations used (Fig. 2), the proliferative response was partly diminished with 1x10 4 macrophages/well for all macrophage sources.This inhibitory effect was especially evident with alveolar macrophages.
For allogeneic MLRs, T lymphocytes isolated from lymph nodes of nonimmunized CBA mice were cultured with C57BL/6 macrophages (1x10 3 , 5x10 3 or 1x10 4 ).A proliferative response by T lymphocytes was obtained (Fig. 3), although increasing the number of macrophages had an inhibitory effect on T cell proliferation.In comparison with other macrophage sources, alveolar macrophages at 1x10 4 macrophage/well exerted the strongest inhibitory effect.
For syngeneic MLRs, macrophages from bone marrow, spleen, peritoneal cavity and alveolus all induced T cell proliferation (Fig. 4).Increasing the number of macrophages in the culture from 1x10 3 to 5x10 3 /well induced proliferation enhancement in three out of the four cases.Lower T cell proliferative responses were obtained with macrophage concentration of 1x10 4 /well.
Thus, an inhibitory effect on T cell proliferation by increasing the number of macrophages was observed both for T lymphoblasts stimulated by antigen and for naïve T cells co-cultured with allogeneic or syngeneic macrophages.In this study we analyzed the ability of tissue specific macrophages to support activation of T lymphoblasts or naïve T cells.It was shown that macrophages isolated from bone marrow, spleen, peritoneal cavity and alveolus supported antigen-specific activation and induced proliferation of allogeneic or syngeneic lymphocytes.The macrophages were isolated by a one-step adherence procedure lasting 24 hours in order to remove dendritic cells which adhere transiently and become non-adherent after 16  SRBC antigens, especially that antigen-specific B cells were necessary at low doses of SRBC ŠMalynn et al., 1985¹, we showed that macrophages isolated from bone marrow, spleen, peritoneal cavity and alveolus could support antigenspecific proliferation of T cells in the presence of high doses (1x10 6 ) of SRBC.As T cells were isolated from lymph nodes of previously immunized mice, we concluded that macrophages are efficient in presenting antigen to T lymphoblasts.
The literature data regarding antigen-presenting ability of macrophages are rather different and dependent on the source of macrophages and the nature of the antigen.It was shown that murine macrophages isolated from the spleen ŠGeijtenbeek et al., 2002¹, as well as bone marrow-derived macrophages ŠLee and Wong, 1982¹ possessed antigen-presenting ability.There are some contradictory data about the functioning of alveolar macrophages as antigenpresenting cells.Depending on animal source as well as the antigen used, alveolar macrophages either support ŠLipscomb et al., 1981¹ or quit ŠFranke-Ullmann et al., 1996¹ antigen-specific T cell proliferation.In contrast to previous reports of either stimulatory or inhibitory effects of lung macrophages on lymphocyte function, Liu et al.Š1984¹ demonstrated that the proliferative response was a complex function of T lymphocyte and macrophage concentrations.They also showed that in the presence of a low concentration of lymphocytes, a low macrophage concentration enhanced proliferation, whereas a higher concentration inhibited proliferation.In the presence of high concentrations of lymphocytes, macrophages only inhibited T cell proliferation.In our experiment, an inhibitory effect on T cell proliferation was obtained.This effect was shown in the presence of all macrophage populations, but was particularly obvious with alveolar macrophages as the antigen-presenting cells.This might be partly explained by the fact that macrophages are a heterogeneous population, even within a particular tissue ŠLaskin et al., 2001¹.It was demonstrated ŠLee and Wong, 1982¹ that only small macrophages induced an enhancement in antigen presentation, whereas large activated macrophages exerted an immunosuppressive effect that probably neutralized any augmentation of stimulatory activity.This inhibitory effect might be a consequence of macrophage heterogeneity, as we have already showed differences in size of 24 In our experimental model macrophages were able to induce allogeneic as well as syngeneic T cell proliferation and an inhibitory effect on T cell proliferation by increasing the number of macrophages was obtained in MLRs.The results presented in this work support the thesis that, depending on the type and number, activation state, interaction and communication with T cells, macrophages could provide either immunostimulatory or immunosuppressive effects on T cells.
Simonovi} Sne`ana et al.Proliferation of naive T lymphocytes and T lymphoblasts in the presence of tissue specific macrophages -hour isolated tissue specific macrophages ŠZivancevic-Simonovic et al., 2004¹.The mixed lymphocyte reaction (MLR) has been used to elucidate naïve T lymphocyte proliferation ŠHart, 1997; Banchereau and Steinman, 1998; Steinman and Witmer, 1978; Massard et al. 1996¹.Although, it is generally accepted that DC stimulate an allogeneic ŠSteinman and Witmer, 1978; Crow and Kunkel, 1982¹ or syngeneic ŠCrow and Kunkel, 1982; Guidos et al., 1984¹ MLR proliferation, the role of macrophages in MLRs is not completely clarified.Therem are data indicating stimulatory ŠMinami et al., 1980¹, enhancing ŠNaito et al., 1989¹ and inhibitory effects ŠSmolen et al., 1981¹ of macrophages on MLRs.