**4. Discussion**

152 Pharmacology

Apart from that function TMf play an important role in their functional contribution of

Similarly to peritoneal macrophages testicular macrophages can be purified by glass adherence or fractionation on discontinuous gradient (Bryniarski 2004). Results in **Table 5** show that TNP substituted TMf (TNP-TMf) when injected i.v. into naïve recipient induce unresponsiveness since the following PCL skin sensitization fail to induce CHS reaction (group B), but TNP-TMf obtained from donors treated with low dose of CY under the seen conditions express strong CHS reaction comparable with control mice actively immunized

> PCL sensitization

A No cells injected + 8.2 1.25 B TNP-TMf + 2.5 0.80 C TNP-TMf (50 mg CY) + 7.5 1.54 Purified TMf obtained from CY-untreated (group B) or CY-treated donors (group B) when substituted with TNP hapten (TNP-TMf) were injected i.v. into mice. Seven days later all TMf recipients and naïve mice (group A) were skin sensitized with 5% PCL and 4 days later challenged on ear skin with 0.4% PCL. 24 h later CHS ear swelling response was measured with engineers micrometer and results expressed in

units of swelling x 10-2mm. Statistics: group A ve group B p<0.001, group B ve group C p<0.01.

Table 5. Testicular macrophages (TMf) from cyclophosphamide-treated mice do not induce

Our further experiments show that the tolerogenic activity of TNP-TMf is mediated by the high cytokine secretory activity mainly TGF-. This cytokine is a very basic tool for functional strategy of TMf within testis and generates the state of organ as immune

The methods used by us for obtaining enriched populations of TMf form mixture of testicular interstitial cells lead to separation of TMf into two functionally different cellular fractions – low density (fractions between interfaces of Percoll gradient 21/27 – 33/39) and high density (over 39/45). Low density fraction produces significantly more TGFthan heavier cells and are CY-sensitive, while high density cells are not (Bryniarski et al. 2004). Our later experiments show that elimination of TGF- activity by injection of anti-TGF mAb, but not anti-IL-10 mAb completely removed unresponsiveness obtained in TNP-TMf recipients after i.v. injection. The other adequate results implementing experiments with

Our experiment with TMf shows that injected intravenously induced CHS response in recipients pre-treated with CY. We found them also actively presenting corpuscular antigen (SRBC) in humoral response (Bryniarski et al. 2004). Again the low density subpopulation of TMf failed to induce CHS when injected i.v. into recipients, but in fact induced the state of

CHS reaction in TMf recipients in units of swelling x 10-2 mm

anatomical blood-testis barrier formed by Sertoli cells (Bryniarski et al 2004).

with PCL (group C ve group A).

privileged site.

Groups Cells injected i.v. before

PCL sensitization

suppressor cells in contrast to non-CY treated donors.

anti-TGF- mAbs are shown in our paper (Bryniarski 2004).

**3.6.1 Induction of contact hypersensitivity by TMf and effect of CY-treatment** 

Our results show that cyclophosphamide in vivo and both its metabolic highly reactive alkylating products --unsaturated aldehyde acrolein (ACR) and nitrogen mustard a derivative of phosphoramide mustard second metabolic agent formed in CY metabolism, activate TNP substituted Mf that leads to activation of CHS reaction mediated by Mf and hapten specific Th1 lymphocytes. We are tempted to suggest that this activity is mediated by the net of different proinflammatory (TNF-, IL-1 IL-6) and suppressory (IL-10 and TGF cytokines secreted by Mf which can uncovering TNP-specific immunization activated by TNP substituted Mf and change their potential from inhibition of unresponsiveness (untreated Mf) into Mf immunogenicity (treated with CY or with CY metabolites).
